Compositions and methods for glucose transport inhibition

ABSTRACT

Glucose deprivation is an attractive strategy in cancer research and treatment. Cancer cells upregulate glucose uptake and metabolism for maintaining accelerated growth and proliferation rates. Specifically blocking these processes is likely to provide new insights to the role of glucose transport and metabolism in tumorigenesis, as well as in apoptosis. As solid tumors outgrow the surrounding vasculature, they encounter microenvironments with a limited supply of nutrients leading to a glucose deprived environment in some regions of the tumor. Cancer cells living in the glucose deprived environment undergo changes to prevent glucose deprivation-induced apoptosis. Knowing how cancer cells evade apoptosis induction is also likely to yield valuable information and knowledge of how to overcome the resistance to apoptosis induction in cancer cells. Disclosed herein are novel anticancer compounds that inhibit basal glucose transport, resulting in tumor suppression and new methods for the study of glucose deprivation in animal cancer research.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Utility patentapplication Ser. No. 15/995,645, filed Jun. 1, 2018, which is acontinuation of U.S. Utility patent application Ser. No. 14/935,902,filed Nov. 9, 2015, now U.S. Pat. No. 10,000,443, which is a acontinuation of U.S. Utility patent application Ser. No. 13/071,386,filed Mar. 24, 2011, now U.S. Pat. No. 9,181,162, which claims priorityto and any other benefit of U.S. Provisional Application No. 61/317,062,filed Mar. 24, 2010, the entire disclosures of which are incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was sponsored in part by the National Science Foundationthrough a Partnership for Innovation Grant (HER-0227907). The UnitedStates government may have certain rights in the invention.

BACKGROUND

Cancer has overtaken cardiovascular diseases as the number one killer inAmerica since 2008 and it was estimated that 565,650 Americans died ofcancer in 2008 alone. Different theories have been proposed for thecause of cancer and numerous strategies have been formulated andexplored for combating the disease. The death rates for some cancerssuch as breast cancer have significantly reduced in the past threedecades primarily due to earlier detection rather than treatments, whilethose of other cancers, such as lung and pancreatic cancer, actuallyincreased. Novel approaches are absolutely and urgently required forfurther improvement in existing cancer therapies and for treating thosecancers for which there are no effective therapies yet. Glucosedeprivation may have the potential to become one such novel andeffective anticancer strategy due to recent progress made inunderstanding of the Warburg Effect, the increased and “addicted”reliance of cancer cells on increased glucose transport and glucosemetabolism, primarily glycolysis.

One of the common features of almost all cancers and also potentiallyone of their common weaknesses is the increased glucose uptake andincreased dependence on glucose as either a source of building blocksfor cell growth and proliferation, a source for energy, or both.Although cancer is not a single disease, different cancers, particularlysolid malignant tumors, do share some common characteristics. One suchcommon characteristic is that they all grow faster than normal cells andhence require more synthetic precursors and more energy to maintaintheir accelerated growth and proliferation rates. Normal cells canutilize different chemicals, such as amino acids, lipids and glucose astheir energy sources.

In contrast to typical cells, the preferred sources for biosynthesismaterials and energy for cancer cells is glucose. For example, healthycolonocytes derive 60-70% of their energy supply from short-chain fattyacids, particularly butyrate. Butyrate is transported across the luminalmembrane of the colonic epithelium via a monocarboxylate transporter,MCT1. Carcinoma samples displaying reduced levels of MCT1 were found toexpress the high affinity glucose transporter, GLUT1, indicating thatthere is a switch from butyrate to glucose as an energy/biosynthesissource in colonic epithelia during transition to malignancy.

The strongest piece of evidence that almost all cancer cells in vivohave increased glucose supply and metabolism as compared with normalcells in the body has been provided by positron emission tomography(PET) scans (FIG. 14). In the PET scan of cancer, ¹⁸F-labeled2-deoxyglucose (2-DG or FDG) as a non-metabolizable glucose analog wasused as a tracer. The regions that light up in the scan are organs,tissues, cells, and cancers that trap more FDG. Brighter spots indicatea higher FDG concentration. This specific PET scan, like many others,reveals that both primary and metastatic cancers (near the lung andarmpit) contain higher FDG concentrations than surrounding normal cells,providing strong evidence that cancer cells have increased glucoseuptake relative to normal cells. The PET scans on various cancer types,including both primary and secondary metastatic cancers, have shown thatalmost all of the studied tumors “trapped” significantly more FDG ascompared to the normal cells and tissues surrounding the tumors.Furthermore, PET scan studies have consistently correlated poorprognosis and increased tumor aggressiveness with increased glucoseuptake and upregulated glucose transporters. Although various theorieshave been proposed to explain the mechanisms by which glucose is usedinside cancer cells, there is a near-unanimous consensus in the fieldthat glucose uptake in almost all malignant tumors is increasedregardless of how glucose is used by cancer cells after it is taken up.The increased glucose uptake and its accompanied increased glucosemetabolism by cancer cells can be, should be, and has been becoming ageneral target for intensive basic and clinical research and fordeveloping novel anti-cancer therapies.

In the 1920s, Warburg discovered that, even in the presence of abundantoxygen, cancer cells prefer to metabolize glucose by glycolysis incytosol than the oxidative phosphorylation in mitochondria as in normalcells. This is seemingly paradoxical as glycolysis is less efficient ingenerating ATP. It has been suggested that such a switch to glycolysisconfers cancer cells some selective advantages for survival andproliferation in the unique tumor microenvironment. Because ofaccelerated growth rates and insufficient oxygen supply, a significantportion of cancer cells in a nodule are in a hypoxic condition, forcingcancer cells to make a shift toward glycolysis by increasing expressionof glucose transporters, glycolytic enzymes, and inhibitors ofmitochondrial metabolism. However, the Warburg Effect cannot beexplained solely by adaptation to hypoxia, since glycolysis is preferredby cancer cells even when ample oxygen is present. Other molecularmechanisms are likely to be involved.

Recent studies have shown that the phenomena observed in Warburg effect,increase glucose consumption and decreased oxidative phosphorylation,and accompanying drastically increased lactate production can also befound in oncogene activation. Ras, when mutated, was found to promoteglycolysis. The activation of Akt was found to increase the rate ofglycolysis, partially due to its ability to promote the expression ofglycolytic enzymes through HIFc. This was speculated as a major factorcontributing to the highly glycolytic nature of cancer cells. Myc, theproto-oncogene and a transcription factor, has also been found toupregulate the expression of various metabolic genes. Tumor suppressors,such as p53, have also been found to be involved in regulation ofmetabolism. All of these recent findings suggest that the Warburg effectin cancer cells is not simply a result of isolated changes in glycolysisalone, but is a biological consequence of extensive communications madethrough known and unknown cross-talk network among multiple signalingpathways. These pathways are involved in cell growth, proliferation, andboth mitochondrial and glucose metabolism that respond to changes inoxygen and nutrient supply. Understanding such extensive signalingnetworks in the Warburg effect is essential for both understanding andcombating cancer.

Some of the most recent studies have focused on glycolytic enzymes,particular on pyruvate kinase (PK). These studies have shown thatincreased glucose transport and glycolysis in cancer cells appear to bedirected toward the generation of building blocks (biosynthesis ofmacromolecules) in cancer cells, and making preparations for celldivision and proliferation rather than as a means to provide bioenergy(ATP). Although aerobic glycolysis is generally accepted as a metabolichallmark of cancer, its causal relationship with tumorigenesis is stillunclear. Glycolysis genes comprise one of the most upregulated gene setsin cancer. Among genes significantly upregulated in tumors is PK, whichregulates the rate-limiting final step of glycolysis. Four isoforms ofPK exist in mammals: the L and R isoforms are expressed in liver and redblood cells; the M1 isoform is expressed in most adult tissues; and theM2 isoform is a splice variant of M1 expressed during embryonicdevelopment. Notably, it has been reported that tumor tissuesexclusively express the embryonic M2 isoform of pyruvate kinase. Becauseof its almost ubiquitous presence in cancer cells, PKM2 has beendesignated as tumor specific, and its presence in human plasma iscurrently being used as a molecular marker for the diagnosis of variouscancers. Both normal proliferating cells and tumor cells express PKM2.PKM2 regulates the proportions of glucose carbons that are channeled tosynthetic processes (inactive dimeric form) or used for glycolyticenergy production (highly active tetrameric form, a component of theglycolytic enzyme complex). In cancer cells, the dimeric form of PKM2 isalways predominant. The switch between the tetrameric and dimeric formof PKM2 allows tumor cells to survive in environments with varyingoxygen and nutrient supplies. The transition between the two formsregulates the glycolytic flux in tumor cells. These findings suggestthat PKM2 is a metabolic sensor which regulates cell proliferation, cellgrowth and apoptotic cell death in a glucose supply-dependent manner.Nuclear translocation of PKM2 was found to be sufficient to induce celldeath that is caspase-independent and isoform-specific. These resultsshow that the tumor marker PKM2 plays a general role incaspase-independent cell death of tumor cells and thereby defines thisglycolytic enzyme as a novel target for cancer therapy development.

Two recent studies demonstrate that PKM2 is regulated by binding tophospho-tyrosine motifs, leading to promotion of increased cell growthand tumor development. PKM2 enhances the use of glycolytic intermediatesfor macromolecular biosynthesis and tumor growth. These findingsillustrate the distinct advantages of this metabolic phenotype in cancercell growth. It appears that the expression of PKM2 and switch fromoxidative phosphorylation to aerobic glycolysis is absolutely requiredfor maintaining cancer growth and proliferation. Thus, inhibitingglycolysis as well as PKM2 may constitute a new and effective anticancerstrategy. These new findings are significant in that they have almostcompletely changed our conventional understanding of the biologicalfunctions of the Warburg effect in cancer, which was believed to be forbiosynthesis of ATP under hypoxic conditions.

Glucose is an essential substrate for metabolism in most cells. Becauseglucose is a polar molecule, transport through biological membranesrequires specific transport proteins. Transport of glucose through theapical membrane of intestinal and kidney epithelial cells depends on thepresence of secondary active Na⁺/glucose symporters, SGLT-1 and SGLT-2,which concentrate glucose inside the cells, using the energy provided byco-transport of Na⁺ ions down their electrochemical gradient.Facilitated diffusion of glucose through the cellular membrane isotherwise catalyzed by glucose carriers (protein symbol GLUT, genesymbol SLC2 for Solute Carrier Family 2) that belong to a superfamily oftransport facilitators (major facilitator superfamily) including organicanion and cation transporters, yeast hexose transporter, planthexose/proton symporters, and bacterial sugar/proton symporters.Molecule movement by such transport proteins occurs by facilitateddiffusion. This characteristic makes these transport proteins energyindependent, unlike active transporters which often require the presenceof ATP to drive their translocation mechanism, and stall if the ATP/ADPratio drops too low.

Basal glucose transporters (GLUTs) function as glucose channels and arerequired for maintaining the basic glucose needs of cells. These GLUTsare constitutively expressed and functional in cells and are notregulated by (or sensitive to) insulin. All cells use both glycolysisand oxidative phosphorylation in mitochondria but rely overwhelmingly onoxidative phosphorylation when oxygen is abundant, switching toglycolysis at times of oxygen deprivation (hypoxia), as it occurs incancer. In glycolysis, glucose is converted to pyruvate and 2 ATPmolecules are generated in the process (FIG. 15). Cancer cells, becauseof their faster proliferation rates, are predominantly in a hypoxic (lowoxygen) state. Therefore, cancer cells use glycolysis (lactateformation) as their predominant glucose metabolism pathway. Such aglycolytic switch not only gives cancer higher potentials for metastasisand invasiveness, but also increases cancer's vulnerability to externalinterference in glycolysis since cancer cells are “addicted” to glucoseand glycolysis. The reduction of basal glucose transport is likely torestrict glucose supply to cancer cells, leading to glucose deprivationthat forces cancer cells to slow down growth or to starve. Thompson'sgroup found that activated Akt led to stimulated aerobic glucosemetabolism in glioblastoma cell lines and that the cells then died whenglucose was withdrawn. This provides direct evidence that cancer cellsare very sensitive to glucose concentration change and glucosedeprivation could induce death in cancer cells.

In normal cells, as shown in FIG. 15, extracellular glucose is taken upby target cells through one or more basal glucose transporters (GLUTs).GLUTs used by cells depend on cell types and physiological needs. Forexample, GLUT1 is responsible for low level of basal glucose transportin all cell types. All GLUT proteins contain 12 transmembrane domainsand transport glucose by facilitating diffusion, an energy-independentprocess. GLUT1 transports glucose into cells probably by alternating itsconformation. According to this model, GLUT1 exposes a singlesubstrate-binding site toward either the outside or the inside of thecell. Binding of glucose to one site triggers a conformational change,releasing glucose to the other side of the membrane. Results oftransgenic and knockout animal studies support an important role forthese transporters in the control of glucose utilization, glucosestorage and glucose sensing. The GLUT proteins differ in their kineticsand are tailored to the needs of the cell types they serve. Althoughmore than one GLUT protein may be expressed by a particular cell type,cancers frequently over express GLUT1, which is a high affinity glucosetransporter, and its expression level is correlated with invasivenessand metastasis potentials of cancers, indicating the importance ofupregulation of glucose transport in cancer cell growth and in theseverity of cancer malignancy. GLUT1 expression was also found to besignificantly higher than that of any other glucose transporters. In onestudy, all 23 tumors tested were GLUT1-positive and GLUT1 was the majorglucose transporter expressed. In addition, both FDG uptake and GLUT1expression appear to be associated with increased tumor size. In severaltumors including NSCLC, colon cancer, bladder cancer, breast cancer, andthyroid cancers, increased GLUT1 expression not only confers a malignantphenotype but also predicts for inferior overall survival. Based on allthese observations, it is conceivable that inhibiting cancer growththrough basal glucose transport inhibition may be an effective way toblock cancer growth and improve on prognosis and survival time.

Evidence indicates that cancer cells are more sensitive to glucosedeprivation than normal cells. Numerous studies strongly suggest thatbasal glucose transport inhibition induces apoptosis and blocks cancercell growth. First, anti-angiogenesis has been shown to be a veryeffective way to restrict cancer growth and cause cancer ablation. Inessence, the anti-angiogenesis approach is to reduce new blood vesselformation and achieve blood vessel normalization inside and surroundingthe tumor nodules. This severely limits the nutrients necessary fortumor growth from reaching the cancer cells. One of the key nutrientsdeprived by anti-angiogenesis is glucose. In this sense, inhibition ofbasal glucose transport can be viewed as an alternative approach toanti-angiogenesis therapy in restricting nutrient supply to cancercells. Thus, the success of the anti-angiogenesis strategy indirectlysupports the potential efficacy of limiting glucose supply to cancercells as a related but novel strategy. Second, inhibitors of variousenzymes involved in glycolysis, have been used to inhibit differentsteps in the glycolysis process, and shown to have significantanti-cancer efficacies. The glycolytic enzymes that have been targetedinclude: hexokinase, an enzyme that catalyzes the first step ofglycolysis; ATP citrate lyase; and more recently pyruvate dehydrogenasekinase (PDK). Among glycolysis inhibitors tested, 3-bromopyruvate and ahexokinase inhibitor were found to completely eradicate advancedglycolytic tumors in treated mice. Compounds targeting mitochondrialglycolytic enzyme lactate dehydrogenase A (LDH-A) have shown significantanti-cancer activity both in vitro and in vivo. This result suggests astrong connection between mitochondrial function and cytosolicglycolysis. 2-DG, the tracer used in PET scans for locating metastasis,has been used as a glucose competitor and a glycolytic inhibitor inanti-cancer clinical trials. These and other related studies have alsoshown that these inhibitors induced apoptosis in cancer cells as acancer cell killing mechanism. Two important conclusions can be drawnfrom all these published studies. (1) The compounds that inhibit varioussteps of glycolysis reduce cancer cell growth both in vitro and in vivo,and (2) inhibiting one of the various steps of glycolysis inducesapoptosis in cancer cells and is an effective anti-cancer strategy. Theyalso strongly suggest that inhibiting glucose transport, the stepimmediately before glycolysis and the first rate-limiting step forglycolysis and all glucose metabolism inside cells, should producebiological consequences to cancer cells similar to or potentially moresevere as glycolysis inhibition. In addition, glucose transport maypotentially be a better target than downstream glycolysis targetsbecause 1) glucose transporters are known to be highly upregulated incancer cells, 2) by restricting the glucose supply at the first step andthus, creating an absolute intracellular glucose shortage, it willprevent any potential intracellular glucose-related compensatory/salvagepathways that cancer cells may use for self-rescue and avoidance of celldeath.

For inhibiting basal glucose transport to become a successfulanti-cancer strategy, it must kill cancer cells without significantlyharming the normal cells. Some experimental observations indicate thatthis is indeed the case. Because cancer cells favor the use of glucoseas the energy source and glycolysis is upregulated in cancer cells,compounds that inhibit glycolysis may kill cancer cells while sparingnormal cells, which can use fatty acids and amino acids as alternativeenergy sources.

It has recently been reported that the addition of anti-GLUT1 antibodiesto various lung and breast cancer cell lines significantly reduced theglucose uptake rate and proliferation of cancer cells, leading toinduction of apoptosis. Furthermore, the antibodies potentiated theanti-cancer effects of cancer drugs such as cisplatin, paclitaxel andgefitinib. These results clearly indicate that agents that inhibitGLUT1-mediated glucose transport are effective either when working aloneor when used in combination with other anti-cancer therapeutics toinhibit cancer cell growth and induce apoptosis in cancer cells. Thesefindings are further supported by two recent publications in whichglucose transport inhibitor fasentin was found to sensitize cancer cellsto undergo apoptosis induced by anticancer drugs cisplatin or paclitaxeland anticancer compound apigenin was found to down-regulate GLUT1 atmRNA and protein levels. Down-regulation of GLUT1 was proposed as thepotential anticancer mechanism for apigenin. All these new findingspoint to the direction that glucose transport inhibitors are likely tosensitize and synergize with other anticancer drugs to further enhanceanticancer efficacy of the drugs. Disclosed herein are compounds andmethods that are 2-5 times more potent than either fasentin or apigeninin inhibiting basal glucose transport and induction of apoptosis.

In one recent study using glucose deprivation, cells growing in highconcentrations of growth factors were found to show an increasedsusceptibility to cell death upon growth factor withdrawal. Thissusceptibility correlated with the magnitude of the change in theglycolytic rate following growth factor withdrawal. To investigatewhether changes in the availability of glycolytic products influencemitochondrion-initiated apoptosis, glycolysis was artificially limitedby manipulating the glucose levels in cell culture media. Like growthfactor withdrawal, glucose limitation resulted in Bax translocation, adecrease in mitochondrial membrane potential, and cytochrome c releaseto the cytosol. In contrast, increasing cell autonomous glucose uptakeby over-expression of GLUT1 significantly delayed apoptosis followinggrowth factor withdrawal. These results suggest that a primary functionof growth factors is to regulate glucose uptake and metabolism and thusmaintain mitochondrial homeostasis and enable anabolic pathways requiredfor cell growth. It was also found that expression of the three genesinvolved in glucose uptake and glycolytic commitment, GLUT1, hexokinase2, and phosphofructokinase 1, was rapidly declined to nearlyundetectable levels following growth factor withdrawal. All of thesestudies suggest that glucose deprivation has been a very valuable andfrequently used method for studying cancer. Intracellular glucosedeprivation can also be created by inhibition of basal glucosetransport. The difference between glucose deprivation resulted fromglucose removal from cell culture media and from inhibition of glucosetransport/glucose metabolism is that glucose removal generates initiallya glucose deprived extracellular environment while inhibition of glucosetransport/glucose metabolism generates a glucose deprived intracellularenvironment without changing or even increasing extracellular glucoseconcentration. The use of glucose transport inhibitors should be able tosupplement and substitute traditional glucose deprivation. Furthermore,traditional glucose deprivation by decreasing extracellular glucoseconcentration cannot be used in animals while inhibitors of glucosetransport can, creating a new approach in studying cancer in vivo and intreating cancer.

SUMMARY

Disclosed herein are compounds of formula (I), in which R₁ is selectedfrom a group consisting of hydrogen, alkyl, benzyl, aryl, and heteroarylmoieties; R₂ is selected from the group consisting of hydrogen, alkyl,benzyl, aryl, heteroaryl, and fluorescent tags; R₃ is selected from thegroup consisting of hydrogen, halo, alkyl, benzyl, aryl, heteroaryl,amino, cyano, and alkoxy; or a salt thereof. In some embodiments, thetwo R₁ groups may be independently selected, and hence different asrecognized by one of skill in the art. In other embodiments, when the R₁groups are different, R₁ may be represents as R₁′ and R₁″ to indicate adifference between R₁ moieties.

In some embodiments, the compound of formula (I) may be further definedto include species where R₁ is an aryl functionality selected from thegroup consisting of 2-, 3-, and 4-hydroxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-,3,4-, and 3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-, and3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl, andperhydroxyphenyl. In other embodiments, the compound of formula (I) maybe further defined where R₂ is a fluorescent tag selected from the groupconsisting of coumarins, dansyl, rhodamine, fluorescein, andcarboxynaphthofluorescein. In some embodiments, the compound of formula(I) is consisting of a molecule, in which R₁ and R₂ are equal to3-hydroxyphenyl and R₃ is a hydrogen atom.

Disclosed herein are compounds of formula (II), in which R₁ is selectedfrom the group consisting of hydrogen, alkyl, benzyl, aryl, andheteroaryl; R₂ is selected from the group consisting of hydrogen, alkyl,benzyl, aryl, and heteroaryl; X is selected from the group consisting ofhydrogen, halo, alkyl, benzyl, aryl, heteroaryl, amino, cyano, andalkoxy; Y is selected from the group consisting of hydrogen, halo,alkyl, benzyl, aryl, heteroaryl, amino, cyano, and alkoxy; or a saltthereof.

In some embodiments, the compound of formula (II) may be further definedto include species where R₁ is an aryl functionality selected from thegroup consisting of 2-, 3-, and 4-hydroxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-,3,4-, and 3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-, and3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl, andperhydroxyphenyl. In other embodiments, the compound of formula (II) isconsisting of a molecule in which R₁ and R₂ are equal to3-hydroxyphenyl, X is equal to fluorine, and Y is equal to hydrogen.

Disclosed herein is a series of compounds of formula (III), in which R₁is selected from the group consisting of hydrogen, halo, alkyl, benzyl,amino, nitro, cyano, and alkoxy; R₂ is selected from the groupconsisting of hydrogen, halo, alkyl, benzyl, amino, nitro, cyano, andalkoxy; R₃ is selected from the group consisting of hydrogen, halo,alkyl, benzyl, amino, nitro, cyano, and alkoxy; X is selected from thegroup consisting of carbon, oxygen, nitrogen and sulfur; and Y isselected from the group consisting of carbon, oxygen, nitrogen andsulfur; or, a salt thereof.

In some embodiments, the compound of formula (III) may be selected fromthe group consisting of the following compounds;

In some embodiments, the compound of formula (III) may be selected fromthe group consisting of the following compounds;

where X is selected from the group consisting of H, 3-Cl, 3-F, 3-CN,4-F, 4-CN, 4-NO₂, 4-SO₂Me, and 4,5-Cl₂. In other embodiments, thecompound of formula (III) is consisting of a molecule in which R₁, R₂,and R₃ are hydrogen, and X and Y are oxygen.

Disclosed herein are compounds of formula (IV), in which R₁ is selectedfrom the group consisting of hydrogen, halo, alkyl, benzyl, amino,nitro, cyano, and alkoxy; R₂ is selected from the group consisting ofhydrogen, alkyl, benzyl, aryl, and heteroaryl; R₃ is selected from thegroup consisting of hydrogen, alkyl, benzyl, aryl, and heteroaryl; or asalt thereof. In some embodiments, the compound of formula (IV) is amolecule, in which R₁ is chlorine, and R₂ and R₃ are2-nitro-5-hydroxyphenyl groups.

Disclosed herein are methods for the treating cancer involving theadministration of a therapeutically effective amount of a compoundselected from the group consisting of formula (I), formula (II), formula(III), and formula (IV) to a subject in need of such treatment.

In some embodiments, the cancer is a solid malignant tumor thatupregulates basal glucose transport via a biological shift fromoxidative phosphorylation to glycolysis in a process known as theWarburg effect. In some embodiments, administration of the compound to ahuman subject may be by any method selected from the group consisting oforal, topical, intra-arterial, intrapleural, intrathecal,intraventricular, subcutaneous, intraperitoneal, intraveneous,intravesicular, and gliadel wafers.

In some embodiments, the compound of formula (I), formula (II), formula(III), and formula (IV) may be administered to a human subject orpatient in combination with one or multiple chemotherapeutic agents as ameans to enhance the efficacy of one or more of the therapeuticallyuseful compounds. In other embodiments, the chemotherapeutic agent thatthe compound of formula (I), formula (II), formula (III), and formula(IV) may be administered in combination with is selected from the groupconsisting of methotrexate, doxorubicin hydrochloride, fluorouracil,everolimus, imiquimod, aldesleukin, alemtuzumab, pemetrexed disodium,palonosetron hydrochloride, chlorambucil, aminolevulinic acid,anastrozole, aprepitant, exemestane, nelarabine, arsenic trioxide,ofatumumab, bevacizumab, azacitidine, bendamustine hydrochloride,bexarotene, bleomycin, bortezomib, cabazitaxel, irinotecanhydrochloride, capecitabine, carboplatin, daunorubicin hydrochloride,cetuximab, cisplatin, cyclophosphamide, clofarabine, ifosfamide,cytarabine, dacarbazine, decitabine, dasatinib, degarelix, denileukindifitox, denosumab, dexrazoxane hydrochloride, docetaxel, rasburicase,epirubicin hydrochloride, oxaliplatin, eltrombopaq olamine, eribulinmesylate, erlotinib hydrochloride, etoposide phosphate, raloxifenehydrochloride, toremifane, fulvestrant, letrozole, filgrastim,fludarabim phosphate, pralatrexate, gefitinib, gemcitabinehydrochloride, gemcitibine-ci splatin, gemtuzumab ozogamicin, imatinibmesylate, trastuzamab, topotecan hydrochloride, ibritumomab tiuxetan,romadepsin, ixabepilone, palifermin, lapatinib ditosylate, lenalidomide,leucovorin calcium, leuprolide acetate, liposomal procarbazinehydrochloride, temozolomide, plerixafor, acetidine, sorafenib tosylate,nilotinib, tamoxifen citrate, romiplostim, paclitaxel, pazopanibhydrochloride, pegaspargase, prednisone, procarbazine hydrochloride,proleukin, rituximab, romidepsin, Talc, sorafenic tosylate, sunitinibmalate, thalidomide, temsirolimus, toremifene, trastuzumub, pantiumumab,vinblastine sulfate, vincristine, vorinostat, and zoledronic acid.

Additional features and advantages will be set forth in part in thedescription that follows, and in part will be obvious from thedescription, or may be learned by practice of the inventions. Theobjects and advantages of the inventions will be realized and attainedby means of the elements and combinations particularly pointed out inthe appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate some embodiments of theinventions, and together with the description, serve to explainprinciples of the inventions.

FIG. 1 shows the initial glucose transport inhibitors 1a and 2a.

FIG. 2 shows glucose uptake results for the possible hydrolyzed productsfrom 1p, 9a, and 2a. These possible hydrolyzed products do not showsignificant inhibition of basal glucose uptake, suggesting that theinhibition was due to the original compounds, not the hydrolyzedproducts.

FIG. 3 shows the molecular structures of α-PGG and β-PGG. PGG has aglucose core that is linked to five galloyl groups through ester bondsthat are formed between the hydroxyl groups of glucose and gallic acids.α-PGG and β-PGG are structural isomers. α-PGG and its derivatives arehydrophilic and are likely to work extracellularly on cell membraneproteins.

FIG. 4 shows the structure of novel inhibitors of basal glucosetransport WZB-25, WZB-26, and WZB-27.

FIG. 5 shows α-PGG treatment induces cell death in cancer cells and thecell death was primarily mediated by apoptosis. A. α-PGG treatment ledto 70-80% reduction of cell viability as measured by a cell viability(MTT) assay. B. α-PGG treatment resulted in more than 2-fold increase inapoptosis in HeLa cells as measured by an apoptosis (ELISA) assay. C.α-PGG treatment of HeLa cells led to a decrease in G1 phase cells but asignificant increase (˜3-fold) in apoptotic cells as determined by anantibody-coupled flow cytometry study.

FIG. 6 shows p53 activation and inactivation as determined by Westernblot analyses. When HeLa, RKO, or MCF-7 cells were treated with 25 μMα-PGG, p53 protein was found not to be activated in HeLa cells but wasactivated in RKO cells.

FIG. 7 shows α-PGG and its derivatives inhibit glucose uptake in HeLa,RKO, and MCF-7 cancer cells. Cells were treated with α-PGG for 20minutes before ³H-labeled 2-DG. Thirty minutes after the addition of2-DG, cells were harvested, lysed, and counted for their respectiveglucose uptake. Samples: 1. Mock; 2. Insulin (100 nM); 3. α-PGG (30 μM);4. WZB-25 (30 μM); 5. WZB-26 (30 μM); and 6. WZB-27 (30 μM).

FIG. 8 shows that α-PGG and its derivatives inhibit basal glucosetransport in HeLa cells in dose-dependent manners. A. α-PGG inhibitsbasal glucose transport in HeLa cells in a dose-dependent manner. B.WZB-25 inhibits basal glucose transport in HeLa cells in adose-dependent manner. C. WZB-27 inhibits basal glucose transport inHeLa cells in a dose-dependent manner.

FIG. 9 shows a time course of glucose transport inhibition induced byα-PGG or α-PGG derivatives. The glucose uptake assay was conducted thesame way as previously described except that the glucose uptake wasterminated at different times (from 1, 5, 10, and up to 30 minutes).

FIG. 10 shows α-PGG induces Akt while α-PGG derivatives do not induceAkt. CHO cells overexpressing the insulin receptor were treated withα-PGG or its derivatives. After treatment, cells were lysed and proteinswere analyzed by the antibody specific for phosphorylated Akt.

FIG. 11 shows PGG-derived compounds induce more cell death in cancercells than in their normal cell counterparts. The left panel showscompounds were used to treat human lung cancer cells (H1299) or normallung cells (NL20) at 25 mM. Forty-eight hours after the treatment, cellviability assay was performed to determine percentage of cell killing.Cells without compound treatment were used as controls (100% baseline).The right panel shows compounds used to treat human breast cancer cells(MCF-7) and normal breast cells (MCF-12A). Cell viability assay wasperformed at the same conditions as for the lung cancer cells in theleft panel.

FIG. 12 shows cell viability and apoptotic assays. A. Cell viabilityassay in RKO colon cancer cells with α-PGG, W25 (WZB-25), W27 (WZB-27),and C7 treatments. RKO cells contain high levels of p53 while RKO E6cells contain much lower levels of p53. B. Apoptosis assay in H1299 lungcancer cells using cleaved PARP protein (89 kDa) as an indicator forCaspase 3 since PARP is a substrate of activated Caspase 3.

FIG. 13 shows a schematic presentation of potential mechanisms forcancer cell killing.

FIG. 14 shows a PET scan of primary and metastatic human cancer.

FIG. 15 shows a schematic presentation of glucose transport in normalcells.

FIG. 16 shows new PGG derivatives.

FIG. 17 shows the structure of two tested compound inhibitors. CompoundsWZB-27 and WZB-115 are polyphenolic compounds derived from PGG. UnlikePGG, they do not have insulin-like glucose uptake stimulatory activity.On the contrary, they only possess potent glucose transport inhibitoryactivity and anticancer activity as demonstrated in glucose uptake andMTT cell viability assays.

FIG. 18 shows cell viability and apoptosis assays (W25=WZB-25,W27=WZB-27). A. Cell viability assay in RKO colon cancer cells. RKOcells contain high levels of p53 while RKO E6 cells contain much lowerlevels of p53. B. α-PGG induces stronger viability-lowering effect inRKO than in RKO-E6 cells. RKO cells have higher level of p53 whileRKO-E6 is p53 deficient. ***p<0.001, **p<0.01. C. Apoptosis assay inA549 lung cancer cells using cleaved PARP protein (89 kDa) as anindicator for Caspase 3 since PARP is a substrate of activated Caspase3. Low glucose (5% of normal) treated samples served as positivecontrols.

FIG. 19 shows cancer cells express more GLUT1 protein and are inhibitedmore by compounds in glucose uptake more their non-cancerouscounterparts. Cancer cells and their non-cancerous counterparts weretreated with or without compounds and then measured for their respectiveglucose uptakes. A. Glucose uptake assay of H1299 lung cancer cells andtheir non-cancerous NL20 cells treated with or without WZB-27. B.Glucose uptake assay of MCF7 cancer cells and their non-cancerous MCF12Acells. C. Western blot analysis of GLUT1 protein expression in cancerand non-cancerous cells using antibody specific against GLUT1 (H43fragment). β-actin served as protein loading control.

FIG. 20 shows blood glucose levels after compound injection. CompoundW27 (=WZB-27) or W115 (=WZB-115) was injected IP into fasting Balb/chealthy mice and blood glucose levels were measured multiple times postinjection. N=5 per group. PBS+DMSO group was the vehicle control.

FIG. 21 shows a combination of glucose inhibitors and anticancer drugsfurther reduces cancer cell viability. Anticancer drugs cisplatin (2.5μM for W27 (=WZB-27) study or 5 μM for W115 (=WZB-115) study) or taxol(2.5 μM) was used to treat either H1299, A549 lung cancer cells or MCF7breast cancer cells in the absence or presence of 10 μM of WZB-115 or 30μM of WZB-27. Cell viability was measured by the MTT assay. The presenceof the compounds significantly increased cancer cell death induced byeither cisplatin or taxol. This experiment was repeated three times andthe results were presented as means±standard deviations.

FIG. 22 shows a comparison study between in house compound inhibitorswith fasentin in glucose uptake inhibition and cell viability in cancercells. Known basal glucose compound fasentin and compound inhibitorsgenerated in house were compared side by side in both glucose uptakeassay and cell viability (MTT) assay. Glucose uptake or cell viabilityof mock treated samples was arbitrarily assigned a value of 100%. A.Glucose uptake assay in H1299 cancer cells. Concentration for allcompounds was 30 μM. B. Cell viability assays in three different cancercell lines. Concentration for all compounds was 60 μM.

FIG. 23 shows an NCI anticancer activity screening results for compoundWZB-115 in 59 cancer cell lines. WZB-115 was sent to NCI for anticanceractivity screening using 59 cancer cell lines, including 9 cancer types.The test was done at a single concentration: 10 μM. Growth rates of mocktreated cancer cells were used as baseline 100%. Any growth rate that issmaller than 100% indicates an inhibition. Because of its promisinganticancer activity profile, NCI has recommended that the compound betested again using five different concentrations to determine its IC₅₀sin these cancer cell lines.

FIG. 24 shows a general scheme for identifying improved basal glucosetransport inhibitors.

FIG. 25 shows several lead compounds and areas for structuralmodification.

FIG. 26 shows an initial set of proposed linkage analogs.

FIG. 27 shows energy-minimized structures of proposed linkage analogs.

FIG. 28 shows biosteric analogs of the phenol group.

FIG. 29 shows an initial set of core aromatic rings.

FIG. 30 shows a comparison of tumor sizes of human lung cancer A549grafted on nude mice. Photos were taken 8 weeks after the treatment.Male NU/J nude mice (7-8 weeks old) were used and purchased from TheJackson Laboratory (Bar Harbor, Me.) and provided the Irradiated TekladGlobal 19% protein rodent diet from Harlan Laboratories (Indianapolis,Ind.). To determine the in vivo efficacy of compound WZB-117 againsthuman NSCLC tumor xenograft growth, exponentially growing A549 cellswere harvested and re-suspended in PBS to achieve a final concentrationof 5×10⁶ cells in 25 μl suspension. Each mouse was injectedsubcutaneously in the right flank with 25 μl cell suspension. At thistime, mice were randomly divided into two groups: control group (n=10)treated with PBS/DMSO (1:1, v/v), and WZB-117 treatment group (n=10),treated with WZB-117 (15 mg/kg). Compound WZB-117 was dissolved inPBS/DMSO (1:1, v/v). Mice were given intraperitoneal injection witheither PBS/DMSO mixture or compound WZB-117 (15 mg/kg) daily since theday of tumor cell inoculation. A. Tumor growth curve. Animal tumor studyindicated that, by daily injection of WZB-117 at the dose of 10 mg/kgbody weight, the tumor size of the compound treated tumors were onaverage approximately 75% smaller than that of the mock treated mice. B.Mouse tumor photos. Tumor-bearing mice on the left were mock-treatedwhile the mice on the right were treated with WZB-117. C. Mouse bodyweight measurements. D. Body weight compositions of the WZB-117 treatedmice compared to those of mock-treated mice.

FIG. 31 shows WZB-117 inhibition of glucose transport by inhibitingGlut1. A. and B. WZB-117 inhibits glucose transport in red blood cells(RBC) in a dose-dependent fashion. C. and D. WZB-117 inhibits glucosetransport in RBC derived “inside out” vesicles (IOV) in a dose-dependentfashion. E. WZB-117 inhibits glucose transport in RBC derived “rightside out” (ROV) vesicles.

FIG. 32 shows WZB-117 treatment of cancer cells induce ER stress,apoptosis and change in glycolytic enzymes. A. WZB-117 treatmentupregulates ER stress protein BiP in a similar way as glucosedeprivation. B. WZB-117 treatment induces cleavage of PARP, suggestingthe apoptosis induction mediated by p53. C. WZB-117 treatmentupregulates the key glycolytic enzyme PKM2 in cancer cells in a similarmanner as the glucose deprivation.

FIG. 33 shows food intake of mice treated with or without WZB-117. Therewas no change of food intake between the PBS/DMSO and the WZB-117treated group during the study. Food intake of each group was measuredevery 7 days since tumor cell inoculation.

FIG. 34 shows blood glucose measurement of tumor-bearing nude micetreated with or without WZB-117. Blood glucose level of each mouse wasmeasured by a blood glucose monitor, and it was measured right beforethe IP injection of WZB-117 (15 mg/kg) and every 30 minutes afterinjection. Food was available to mice during the measurement. Data wasexpressed in average+standard deviation. No significant difference inblood glucose levels was found between untreated and compound WZB-117injection group immediately after the compound injection or during orafter the animal study.

FIG. 35 shows that compound WZB-117 kills significantly more cancercells than non-cancerous cells. A. A549 lung cancer and B. MCF7 breastcancer cells were treated with or without WZB-117 for 48 hr, and thenmeasured for their respective viability rates with the MTT assays.Mock-treated cells served as controls (100% viability) for comparison.Noncancerous NL20 and MCF12A cells were treated the same way forcomparison.

FIG. 36 shows the anticancer activity of WZB-117 as demonstrated byclonogenic assays. Three cancer cell lines A549, H1299 (lung cancers)and MCF7 (breast cancer) grown in culture dishes were treated withWZB-117 or a weaker inhibitor WZB-134 or no compound (mock) for 48 hrs.Then the treated cells were allowed to grow back in compound-free normalcell culture medium for 2 weeks and then stained with crustal violet andcounted for number of survived clones. The fewer and smaller the stainedspots (clones), the higher the inhibition.

FIG. 37 shows the structures of several novel glucose transportinhibitors WZB-115, 117, and 173. Compound 117 is an analog of 115 while173 is an ether-bond analog. Compounds WZB-117 and WBZ-173 are derivedfrom compound WZB-115, which is a polyphenolic model compound used inour previous cancer studies. WZB-115 was derived from a naturalanticancer and antidiabetic compound called penta-galloyl-glucose (PGG).WZB-117 and WZB-173 are very similar structurally to 115 but are bothstructurally simplified and functionally optimized compared to WZB-115.As a result, WZB-117 and WZB-173 are more potent in their anticanceractivities than WZB-115 and are also structurally more stable than 115in solution and cell culture media.

DETAILED DESCRIPTION

The present inventions will now be described by reference to some moredetailed embodiments, with occasional reference to the accompanyingdrawings. These inventions may, however, be embodied in different formsand should not be construed as limited to the embodiments set forthherein. Rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of theinventions to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these inventions belong. The terminology used in thedescription of the inventions herein is for describing particularembodiments only and is not intended to be limiting of the inventions.As used in the description of the inventions and the appended claims,the singular forms “a,” “an,” and “the” are intended to include theplural forms as well, unless the context clearly indicates otherwise.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present inventions. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the inventions are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Every numerical range given throughoutthis specification will include every narrower numerical range thatfalls within such broader numerical range, as if such narrower numericalranges were all expressly written herein.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Definitions

“Alkyl” shall refer to any chemical compound that consists only of theelements hydrogen and carbon, wherein the atoms are linked togetherexclusively through single bonds. The term alkyl may also be extended tomean any chemical compound that consists only of the elements hydrogen,fluorine, and carbon, wherein the atoms are linked together exclusivelythrough single bonds. This class of fluorinated compounds may also bereferred to as “fluoroalkanes,” “fluoroalkyl,” “fluoroalkyl groups,” and“fluorocarbons.” Examples of hydrocarbons include, but are not limitedto methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, sec-butyl,i-butyl, t-butyl, cyclobutyl, n-pentyl, i-pentyl, neo-pentyl,cyclopentyl, n-hexyl, cyclohexyl, thexyl, n-heptyl, n-octyl, n-decyl,and adamantyl. Examples of fluorinated compounds include, but are notlimited to fluoromethyl, difluoromethyl, trifluoromethyl,1,1,1-trifluoroethyl, 2,2-difluoroethyl, and perfluoroethyl.

“Benzyl” shall be used to describe the substituent or molecular fragmentpossessing a structure related to RC₆H₄CH₂— of an organic compound. Asubstituted benzyl compound may also be described as any aryl orheteroaryl ring system attached to a methylene (—CH₂—) subunit. Examplesof benzyl groups include, but are not limited to benzyl; 2, 3, and4-halobenzyl; 2, 3, and 4-alkylbenzyl; 2, 3, and 4-cyanobenzyl; 2, 3,and 4-ketobenzyl; 2, 3, and 4-carboxybenzyl; 2, 3, and 4-aminobenzyl; 2,3, and 4-nitrobenzyl; 2, 3, and 4-hydroxybenzyl; 2, 3, and4-alkoxybenzyl; disubstituted benzyl, and trisubstituted benzylderivatives.

“Aryl” shall mean any functional group on a compound that is derivedfrom a simple aromatic ring. Examples include, but are not limited tophenyl; 2-, 3-, and 4-hydroxyphenyl; 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and3,5-dihydroxyphenyl; 2,3,4-, 2,3,5-, 2,3,6-, and 3,4,5-trihydroxyphenyl;2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl; perhydroxyphenyl; 2, 3, and4-halophenyl; 2, 3, and 4-alkylphenyl; 2, 3, and 4-cyanophenyl; 2, 3,and 4-ketophenyl; 2, 3, and 4-carboxyphenyl; 2, 3, and 4-aminophenyl; 2,3, and 4-nitrophenyl; 2, 3, and 4-hydroxyphenyl; 2, 3, and4-alkoxyphenyl; disubstituted phenyl, and trisubstituted phenylderivatives.

“Heteroaryl” shall mean any functional group on a compound that isderived from a heteroaromatic ring. Heteroaromatic species contain aheteroatom, or an atom other than hydrogen or carbon, including, oxygen,nitrogen, sulfur, phosphorous, silicon, and boron. Examples include, butare not limited to furans, benzofurans, thiophenes, benzothiophenes,pyrroles, indoles, and borabenzenes.

“Halo” and “halogen” shall refer to any element of the periodic tablefrom Group 17, consisting of fluorine, chlorine, bromine, iodine, andastatine.

“Amine” and “amino” shall refer to any organic compound or functionalgroup that contains a basic nitrogen with a lone pair of electrons.Amines are derived from ammonia and may be primary, secondary, andtertiary. Examples of amines include, but are not limited to ammonia,methylamine, dimethlamine, trimethylamine, ethylamine, diethlamine,triethylamine, ethyldimethylamine, isopropylamine, diisopropylamine,diisopropylethylamine, diphenylamine, dibenzylamine, t-butylamine,analine, and pyridine.

“Cyano” shall be considered synonymous with the organic functionalitynitrile, which contains a carbon triple-bonded to a nitrogen atom.Cyanides tend to be highly toxic in nature, and are typically found assalts.

“Alkoxy” shall mean an alkyl group singly bonded to an oxygen. The rangeof alkoxy groups is great, ranging from methoxy to any number ofarylalkoxy groups. Examples of alkoxy groups include, but are notlimited to methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, t-butoxy,and phenoxy.

“Fluorescent tags,” “fluorescent molecule,” “fluorophore,” and“fluorescent labels” shall mean any portion of a molecule thatscientists or researchers have attached chemically to aid in thedetection of the molecule to which it has been attached. Examples offluorescent tags include, but are not limited to coumarins, dansyl,rhodamine, fluorescein, carboxynaphthofluorescein, and fluorescentproteins.

A “salt” shall refer to an ionic species resulting from the pairing ofan anionic derivative of one of the compounds from formulas 1, 2, 3, and4 with a cationic species. The cationic species may include, but is notlimited to lithium, sodium, potassium, rubidium, cesium, beryllium,magnesium, calcium, strontium, barium, aluminum, copper, zinc, iron,chromium, manganese, nickel, palladium, platinum, indium, rhodium, andarsenic.

“Therapeutically effective” when used to describe an amount of acompound applied in a method, refers to the amount of a compound thatachieves the desired biological effect, for example, an amount thatleads to the inhibition of basal glucose transport.

“Inhibit” or “stop” shall mean reduce, inhibit, damage, eliminate, kill,or a combination thereof.

“Lowering” in the context of basal glucose transport shall mean toreduce the efficiency of glucose transport within a cancer cell.

Structure of α-PGG-Derived Generation 1 Compounds; WZB-25, WZB-26, andWZB-27

The methods for the synthesis of compounds WZB-25, WZB-26, and WZB-27are as follows:

Acid chloride 2 (863 mg, 1.88 mmol) was added to a solution of1,6-anhydro-β-D-glucose (100 mg, 0.62 mmol) in anhydrous acetonitrile(25 mL) at room temperature. DMAP (241 mg, 1.97 mmol) was added to thereaction mixture after stirred for 30 min at room temperature, themixture was stirred for 24h and the solvent was removed, the crude waspurified by chromatography on silica gel giving 730 mg of 3 in 83%yield. ¹H NMR (CDCl3) δ 7.60-7.31 (m, 51H), 5.88 (s, 1H), 5.69 (t, 1H,J=3.0 Hz), 5.29 (d, 3H, J=5.3 Hz), 5.23 (s, 4H), 5.11-5.02 (m, 13H),4.98 (d, 1H, J=6.2 Hz), 4.28 (d, 1H, J=7.4 Hz), 3.97 (q, 1H, J=6.2, 7.4HZ).

10% palladium on carbon (21 mg, 0.02 mmol) was added to a solution of 3(420 mg, 0.29 mmol) in anhydrous THF (20.0 mL), the mixture was stirredunder hydrogen gas atmosphere for overnight at room temperature. Themixture was filtered through Celite, the filtrate was diluted withmethanol and dichloromethane and filtered through Celite three timesuntil the solution was clear. The solvent was removed and gave crude 4in 64% yield.

Acid chloride 2 (941 mg, 2.05 mmol) was added to a solution of3-methoxycatechol (140 mg, 1.00 mmol) in anhydrous acetonitrile (10 mL)at room temperature. DMAP (268 mg, 2.20 mmol) was added to the reactionmixture after stirred for 30 min at room temperature, the mixture wasstirred for 2 days and the solvent was removed, the crude was purifiedby chromatography on silica gel (25% EA in hexane) giving 708 mg of 6 in72% yield. ¹H NMR (CDCl3) δ 7.64 (d, 4H, J=16.7 Hz), 7.51-7.33 (m, 31H),7.16 (d, 1H, J=8.2 Hz), 7.06 (d, 1H, J=8.4 Hz), 5.18 (d, 4H, J=7.0 Hz),5.06 (d, 8H, J=11 Hz), 3.96 (s, 3H); ¹³C NMR (CDCl3) δ 164.0, 163.6,153.1, 152.8, 152.3, 144.1, 143.2, 137.6, 137.5, 136.5, 136.4, 132.3,128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7, 126.6, 123.9,123.8, 115.4, 110.2, 109.7, 109.6, 75.3, 71.2, 56.4.

10% palladium on carbon (43 mg, 0.04 mmol) was added to a solution of 6(500 mg, 0.51 mmol) in anhydrous THF (20.0 mL), the mixture was stirredunder hydrogen gas atmosphere for 12h at room temperature. Then themixture was filtered through Celite, the filtrate was concentrated andpurified by chromatography on silica gel giving 9.5 mg of 7. Most ofcompound 7 was decomposed on silica gel. ¹H NMR (CDCl3) δ 8.30 (brs,6H), 7.31-7.13 (m, 5H), 7.04 (d, 1H, J=8.1 Hz), 6.95 (d, 1H, J=8.3 Hz),3.83 (s, 3H).

Acid chloride 2 (1.40 g, 3.05 mmol) was added to a solution ofpyrogallol (126 mg, 1.00 mmol) in anhydrous acetonitrile (15 mL) at roomtemperature. DMAP (391 mg, 3.20 mmol) was added to the reaction mixtureafter stirred for 30 min at room temperature, the mixture was stirredfor 24h and the solvent was removed, the crude was purified bychromatography on silica gel (25% EA in hexane as eluent) giving 570 mgof 9 in 41% yield. ¹H NMR (CDCl3) δ 7.53 (s, 4H), 7.49-7.20 (m, 50H),5.10 (s, 4H), 5.01 (s, 8H), 4.94 (s, 2H), 4.85 (s, 4H); ¹³C NMR (CDCl3)δ 163.6, 163.0, 152.7, 144.2, 143.6, 143.3, 137.4, 137.3, 136.3, 136.1,135.2, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 126.3,123.6, 123.1, 120.9, 109.6, 75.2, 75.1, 71.2.

10% palladium on carbon (16 mg, 0.024 mmol) was added to a solution of 6(260 mg, 0.29 mmol) in anhydrous THF (15.0 mL), the mixture was stirredunder hydrogen gas atmosphere for overnight at room temperature. Thenthe mixture was filtered through Celite, the filtrate was concentratedand purified by chromatography on silica gel (25% EA in hexane) giving11.3 mg of 10. Compound 10 was decomposed on the column. ¹H NMR (CDCl3)δ 8.24 (brs, 4H), 7.47-7.41 (m, 1H), 7.34-7.32 (m, 2H), 7.17 (s, 4H),7.09 (s, 2H), 2.92 (brs, 5H).

Evaluation of Generation 1 Compounds Derived from α-PPG

Compounds 1a and 2a were initially prepared as potential anti-diabeticanalogs of α-PGG (FIG. 1). Given the tight SAR of this class ofcompounds, it was hypothesized that a more rigid scaffold (i.e., abenzene ring) might enhance the activity. Rather than possessinginsulin-like activity, these two compounds were surprisingly andserendipitously found to inhibit basal glucose transport on cervical(HeLa), colon (RKO), and breast (MCF-7) cancer cells at a concentrationof 30 μM (Table 1).

TABLE 1 Glucose transport inhibitory activity (%) of 1a and 2a indifferent cancer cell lines Compound Hela RKO MCF-7 1a 46.3 ± 3.3 57.2 ±4.4 33.0 ± 0.5 2a 36.0 ± 4.9 58.1 ± 0.1 60.4 ± 2.4

Compounds 1a and 2a also inhibited basal glucose transport in H1299cells by 58.4±6.3% and 86.1±1.0%, respectively (Table 2), as measured bya standard glucose uptake assay compared to non-compound treated cellscontrols (considered as 0% inhibition). Tested in an MTT cellproliferation assay in H1299 cells, their inhibitory activities oncancer cell growth were found to be 36.0±6.1% and 39.9±5.0%,respectively (non-compound treated cell controls were considered as 0%inhibition).

Given the potential utility of the inhibition of glucose transport fordevelopment of novel anticancer agents, structure-activity relationshipof these compounds as both inhibitors of glucose transport and cancercell proliferation were investigated. Based on these two compounds anumber of derivatives were prepared in order to understand the need forthe trihydroxyphenyl ester and the need for three of these esters on thecentral aromatic ring.

The desired analogs were prepared by the acylation of a series of di-and tri-hydroxy benzenes with a group of substituted benzoyl halides. Agroup of mono-, di-, and trihydroxybenzoyl halides as well asmethoxybenzoyl halides were chosen as acylating agents. The synthesis ofthe hydroxybenzoyl halides is outlined in Scheme 1. Commerciallyavailable phenols 3a-f were perbenzylated and the resulting estershydrolyzed and the acid converted to the acid chlorides 5a andbenzyloxybenzoyl chlorides 5b, 5c, 5d, 5e, and 5f. The requisitemethoxy-substituted benzoyl halides were prepared from the commerciallyavailable carboxylic acids.

TABLE 2 Compounds prepared, their induced inhibitory activities in basalglucose transport and cell growth in H1299 lung cancer cells GlucoseCell Com- transport growth pound Yield^(a) inhibition^(b) inhibition^(b)# Ar/Ar′ X Y (%) (%) (%) 1a 3,4,5-(OH)₃—C₆H₂ OMe H 69  58.4 ± 1.0^(c)36.0 ± 6.1 2a 3,4,5-(OH)₃—C₆H₂ — — 78 86.1 ± 1.0 39.9 ± 5.0 1b3,4,5-(OH)₃—C₆H₂ H Cl 69 84.4 ± 0.1 — 1c 3,4,5-(OH)₃—C₆H₂ F H 78 81.1 ±1.1 — 1d 3,4,5-(OH)₃—C₆H₂ H H 65 32.1 ± 6.2 — 9a 3,4,5-(OMe)₃—C₆H₂ — —86 41.1 ± 1.5 — 7a 3,4,5-(OMe)₃—C₆H₂ OMe H 90 33.3 ± 3.6 22.2 ± 3.4 7h3,4,5-(OMe)₃—C₆H₂ H Cl 89  1.7 ± 1.1 20.6 ± 2.1 7c 3,4,5-(OMe)₃—C₆H₂ F H87  7.9 ± 2.6 19.6 ± 3.6 9b 2,6-(OMe)₂—C₆H₃ — — 69 75.8 ± 4.2 — 9c3,4-(OMe)₂—C₆H₃ — — 78   0 ± 3.9 10.0 ± 1.1 9d 3-(OMe)—C₆H₄ — — 92 66.2± 1.8 — 7d 3-(OMe)—C₆H₄ OMe H 96 32.1 ± 0.2 18.3 ± 4.3 2b 3,5-(OH)₂—C₆H₃— — 81 98.7 ± 0.8 41.0 ± 5.5 1e 3,5-(OH)₂—C₆H₃ OMe H 78 69.4 ± 3.0 — 1f3,5-(OH)₂—C₆H₃ H Cl 87 95.2 ± 0.2 38.8 ± 6.9 1g 3,5-(OH)₂—C₆H₃ F H 8194.0 ± 0.8 35.0 ± 7.6 2c 3,4-(OH)₂—C₆H₃ — — 81 94.7 ± 0.4 42.2 ± 6.1 1h3,4-(OH)₂—C₆H₃ OMe H 78 0 32.4 ± 2.7 1i 3,4-(OH)₂—C₆H₃ H Cl 87 94.4 ±0.4 45.2 ± 7.6 1j 3,4-(OH)₂—C₆H₃ F H 81 88.7 ± 1.8 41.5 ± 5.5 2d2-(OH)—C₆H₄ — — 88 74.7 ± 2.0 — 1k 2-(OH)—C₆H₄ OMe H 91 50.5 ± 7.6 18.0± 4.4 1l 2-(OH)—C₆H₄ H Cl 92 51.6 ± 5.9 26.6 ± 2.3 1m 2-(OH)—C₆H₄ F H 8951.0 ± 6.6 — 2e 4-(OH)—C₆H₄ — — 81 88.7 ± 2.5 34.8 ± 7.7 1n 4-(OH)—C₆H₄OMe H 78 86.5 ± 2.8 36.6 ± 6.7 1o 4-(OH)—C₆H₄ H Cl 87 79.0 ± 8.7 — 1p4-(OH)—C₆H₄ F H 84 92.6 ± 0.7 35.9 ± 4.7 1q 4-(OH)—C₆H₄ H H 82 57.5 ±2.9 — 2f 3-(OH)—C₆H₄ — — 81 99.7 ± 0.1 59.3 ± 4.9 1r 3-(OH)—C₆H₄ OMe H78 79.0 ± 8.7 — 1s 3-(OH)—C₆H₄ H Cl 87 93.1 ± 1.7 44.5 ± 5.2 1t3-(OH)—C₆H₄ F H 81 92.8 ± 0.1 40.8 ± 5.6 ^(a)Overall yield from 6 to 8.^(b)Untreated cells served as negative controls (0% inhibition).^(c)Data were presented as mean ± standard deviation.

Scheme 1. Synthesis of the Benzyloxybenzoyl Chlorides 5

In terms of the core aromatic ring, pyrogallol 8 (present in 2a) and3-methoxycatechol 6a (X═OMe, Y═H, present in 1a) were chosen. Twohalogen substituted phenols, 6b (X═H, Y═Cl) and 6c (X═F, Y═H) werechosen to provide a p-donor (similar to the methoxy group) but electronwithdrawing group. An unsubstituted catechol 6d (X, Y═H) was included.As shown in Scheme 2, each of these phenols was then coupled to each ofthe acid chlorides 5a-f and several methoxy-substituted benzoylchlorides. After coupling, the benzyloxy esters were deprotected viacatalytic hydrogenation. The overall yields are shown in Table 2.

As shown in Table 2, compounds were tested in a standard glucose uptakeassay. Briefly, H1299 cancer cells were treated with or without thecompounds (30 μM) in triplicates for 10 min before the glucose uptakeassay. Cellular glucose uptake was measured by incubating cells in theglucose-free KRP buffer with 0.2 Ci/mL [³H]2-deoxyglucose (specificactivity, 40 Ci/mmol) for 30 min in the absence or presence ofcompounds. After the cells were washed with ice-cold PBS and lysed by0.2 N NaOH, the cell lysates were transferred to scintillation countingvials and the radioactivity in the cell lysates was quantified by liquidscintillation counting. Cell growth measurements were performed using anMTT assay in hexads in a 96-well tissue culture plate with 5,000 cellsplated in each well. The cells were incubated in presence or absence ofthe compounds (30 μM) for 48 h. After incubation, cell viabilities wereassayed using a 96-well SPECTRAMAX™ absorbance/fluorescence plate reader(Molecular Devices).

In some embodiments, comparison of compounds 1a and 2a to derivatives inwhich the core aromatic ring was substituted with a fluorine or chlorine(1b and 1c), revealed that both of these compounds have similar activityin the glucose transport inhibition assay as compound 2a and weresignificantly better than 1a. Clearly the halogen substitution on thecore aromatic ring is important. An unsubstituted core aromatic ring Idshowed lower levels of inhibition relative to 1a and 2a. In order todetermine the necessity of the phenolic hydroxyl groups we preparedseveral derivatives of 1a and 2a in which the OH group was replaced witha methoxyl group (9a-d, 7a-d). These compounds showed uniformly lowerlevels of inhibition of glucose transport. Only compounds 9b and 9d(2,6-dimethoxybenzoyl and 3-methoxybenzoyl) showed moderate levels ofinhibition.

In some embodiments, the phenolic hydroxyl groups were systematicallyremoved preparing a series of di-hydroxyl and mono-hydroxyl derivatives.The 3,5-dihydroxy and 3,4-dihydroxyl derivatives overall showed goodlevels of glucose transport inhibition. In both series the tribenzoylderivatives (2b and 2c) as well as the fluoro- and chloro-substitutedderivatives showed >90% inhibition while the methoxy-substitutedderivatives (1e and 1h) showed little to no inhibition. All of thecompounds showing >90% glucose transport inhibition also show ˜40%decrease in cancer cell growth rate.

In some embodiments, removing an additional hydroxyl group provided aset of monohydroxyl compounds at the 2-, 3-, and 4-positions. The2-hydroxyl series showed uniformly poorer inhibition of glucosetransport. The 4-hydroxyl series while showing poorer inhibition ofglucose transport did provide compounds with >85% inhibition of glucosetransport. These compounds showed a >35% decrease in cancer cell growthrate. The 3-hydroxyl series showed excellent inhibition of glucosetransport with the tribenzoyl derivative 2f showing >99% inhibition ofglucose transport. This compound also showed the highest level of cellgrowth inhibition at ˜60%. By analyzing all data in Table 2, it wasfound that the linear correlation coefficient R=0.817 (R2=0.667),indicating that approximately ⅔ (66.7%) of the inhibitory activity ofcancer cell growth came from the inhibitory activity of basal glucosetransport.

In general, the presence of a hydroxy group at the 3-position of thependant benzoyl group is important for both inhibitions of glucosetransport and cancer cell growth. This 3-hydroxy group can be attachedto a chloro- or fluoro-substituted core benzene ring or be part of atribenzoyl system. Unsubstituted or electron-donating substituents leadto significant decreases in the inhibition of glucose transportactivity.

In other embodiments, the hydrolysis products (8, 6c, 10, 11, and 12) ofthe phenolic esters may exhibit glucose uptake inhibition activity. Asshown in FIG. 2, none of these compounds had any glucose uptakeinhibition activity. Similarly, none of these compounds showed anyactivity in anticancer screens. In summary, a library of multiphenolicester compounds as novel inhibitors of basal glucose transport andanticancer agents with a potential new target, basal glucose transport,were synthesized.

Potent and Selective Inhibitors of Basal Glucose Transport have beenIdentified Through an SAR Study

During a structure activity relationship (SAR) study in which more than80 PGG analogs were synthesized and analyzed by glucose uptake assay andfunctional assays, a group of inhibitors for glucose uptake, which hadan opposite activity as PGG, were also identified. It wasserendipitously discovered that these inhibitors caused inhibition ofcancer cell growth and cancer cell death. With these new findings, itwas decided to chemically synthesize more potent and selectiveinhibitors and use them for cancer study.

Given the tight SAR of the initial set of PGG analogs, it washypothesized that a more rigid scaffold upon which the galloyl group wasappended might provide enhanced potency and selectivity. Several newanalogs of the PGG class of compounds were prepared. Two compounds werebased upon an aromatic nucleus (as opposed to the glucose nucleus). Thehypothesis for all compounds was that the rigid central core wouldprevent any conformational mobility associated with the glucose nucleus.When these compounds (WZB-26 and WZB-27) were assayed, it was found thatthey possessed basal glucose transport inhibitory activity without anyinsulin-like activity. Thus, these compounds are selective inhibitors ofbasal glucose transport of animal cells. When these compounds were usedto treat different cancer cell lines, they were found to also inhibitbasal glucose transport in cervical (HeLa), colon (RKO), and breast(MCF7) cancer cells. This result suggests that these compounds aregeneral basal glucose transport inhibitors; inhibiting all three cancercell lines tested. Based on these two compounds a number of derivativeswere prepared in order to understand the need for the trihydroxyphenylester and the need for three of these esters on the central aromaticring (FIG. 16). In brief, a series of di- or trihydroxybenzenes wereacylated with a protected hydroxybenzoic acid. After coupling theprotecting group (benzyl) was removed to provide the target compounds,either a tribenzoyl derivative (3) or a phenyl substituted dibenzoylderivative (5).

Compounds WZB-26 and WZB-27 were the first compounds prepared. Bothcompounds showed similar inhibition of basal glucose transport (˜85%).Initially, the substitution pattern on the central aromatic ring was ofinterest. As WZB-27 has three galloyl group while WZB-26 has only 2 butan additional methoxy group. Preparation of multiple compounds wascompleted to explore the impact other substituents on the centralaromatic ring would have upon the levels of inhibition. In someembodiments, the compounds prepared may be designated WZB-89 (Fsubstitution), WZB-90 (Cl substitution), and WZB-110 (H substitution).Both WZB-89 and WZB-90 showed similar levels of activity to WZB-26 and-27 while the unsubstituted WZB-101 showed dramatically decreased levelsof activity. Clearly some type of it-donor group in the 3- or 4-positionas seen in WZB-26, -27, -89, and -90 is beneficial to activity. BothWZB-26 and WZB-27 have three phenolic hydroxyl groups. In order todetermine if these hydroxyl groups were acting as H-bond donors orH-bond acceptors, 4 analogs in which the OH group was replaced with anOMe group (WZB-76, -81, -101, -102) were prepared. The activity of thesecompounds was dramatically decreased, clearly indicating the need for anH-bond donor (i.e. a phenolic OH). Next, the need for the galloyl group(i.e. 3,4,5-trihydroxybenzoyl) was examined by a systematic removal ofthe hydroxy group. A series of dihydroxy (3,5-dihydroxy and3,4-dihydroxy) and monohydroxy (2-OH, 3-OH, and 4-OH) derivatives wereprepared. In all cases analogs with a methoxy group or no substitutionon the central aromatic ring provided significantly lower levels ofinhibition regardless of the number or position of hydroxyl groups onthe pendant benzoyl ester. Of the analogs prepared the 3,5-dihydroxy(WZB-111, WZB-113, WZB-114), the 3,4-dihydroxy (WZB-119, WZB-121,WZB-112) and the 3-monohydroxy (WZB-115, WZB-117, WZB-118) derivativesshowed inhibition levels of 95-99%. As there is an interest in thepharmaceutical industry to develop simpler, lower molecular weightinhibitors, compounds WZB-27 and WZB-115 were used for biologicalstudies first and better compounds would be employed in later synthesisand assays. Table 3 indicates that we have systematically synthesizedmore than 100 different compounds to study SAR of the compounds with anobjective of making more potent and selective inhibitors to basalglucose transport. As shown in Table 3, some of these inhibitors are thebest basal glucose transport inhibitors reported to date and may beuseful for future clinical studies.

TABLE 3 Analogs prepared and basal glucose transport inhibition Compound# X Y (OR)_(n) % inhibition^(a) WZB-26 OMe H 3,4,5-(OH)₃ 84.0 ± 1.9WZB-27 NA, tribenzoyl analog 3 3,4,5-(OH)₃ 86.1 ± 1.0 WZB-89 F H3,4,5-(OH)₃ 81.0 ± 1.1 WZB-90 H Cl 3,4,5-(OH)₃ 84.4 ± 0.1 WZB-110 H H3,4,5-(OH)₃ 32.1 ± 6.2 WZB-76 NA, tribenzoyl analog 3 3,4,5-(OMe)₃ 41.1± 1.5 WZB-81 OMe H 3,4,5-(OMe)₃ 33.3 ± 3.6 WZB-101 F H 3,4,5-(OMe)₃  7.9± 2.6 WZB-102 H Cl 3,4,5-(OMe)₃  1.7 ± 1.1 WZB-111 NA, tribenzoyl analog3 3,5-(OH)₂ 99.5 ± 0.3 WZB-112 OMe H 3,5-(OH)₂ 69.4 ± 3.0 WZB-112 OMe H3,5-(OH)₂ 69.4 ± 3.0 WZB-113 F H 3,5-(OH)₂ 96.7 ± 1.6 WZB-114 H Cl3,5-(OH)₂ 96.1 ± 0.1 WZB-119 NA, tribenzoyl analog 3 3,4-(OH)₂ 94.7 ±0.4 WZB-120 OMe H 3,4-(OH)₂ 0 WZB-121 F H 3,4-(OH)₂ 88.7 ± 1.8 WZB-122 HCl 3,4-(OH)₂ 94.4 ± 0.4 WZB-91 NA, tribenzoyl analog 3 4-OH 88.7 ± 2.5WZB-92 F H 4-OH 92.6 ± 0.7 WZB-93 OMe H 4-OH 86.5 ± 2.8 WZB-94 H Cl 4-OH79.0 ± 8.7 WZB-103 H H 4-OH 57.5 ± 2.9 WZB-115 NA, tribenzoyl analog 33-OH 99.7 ± 0.1 WZB-116 OMe H 3-OH 79.0 ± 8.0 WZB-117 F H 3-OH 98.3 ±8.0 WZB-118 H Cl 3-OH 96.5 ± 0.4 WZB-127 NA, tribenzoyl analog 3 2-OH74.7 ± 2.0 WZB-128 OMe H 2-OH 50.5 ± 7.6 WZB-129 F H 2-OH 51.0 ± 6.6WZB-130 Cl H 2-OH 51.6 ± 5.9 ^(a)Compounds were tested at 30 μM in H1299cells by the glucose uptake assay. Cells without being treated by anycompound served as negative controls (0% inhibition).

α-PGG Induces Apoptosis in Human Colon, Cervical, and Breast CancerCells

When α-PGG was used to treat RKO (colon), HeLa (cervical), and MCF-7(breast) human cancer cells, it was found that the treatment resulted inpronounced cell death (FIG. 5A), and the cell death was causedprimarily, if not exclusively, by apoptosis (FIGS. 5B & 5C). α-PGG doesnot cause much apoptosis in normal (non-cancer) cells (data not shown),indicating the compound shows increased cytotoxicity more towards cancercells.

PGG-Derived Compounds Induce Cell Death Preferentially in Cancer Cellsthan their Normal Counterparts

Cancer cells heavily depend on glucose as their preferred energy sourceand glucose deprivation has been proposed as an anti-cancer strategy. Inorder for these compounds to be effective anti-cancer agents, they mustbe able to kill more cancer cells than normal cells. Cell killing (orcell viability) assays revealed that some of these compounds,particularly WZB-27 (=W27), preferentially kill cancer cells (NSCLCH1299 and breast carcinoma MCF7) than their non-cancerous cellcounterparts (NL20 or MCF12A cells, Table 3). These results suggest thatthese compounds have excellent potential to be anti-cancer agents. Basedon this observation, it was speculated that optimal compounds and drugconcentration can be determined that will minimally impact normal cellswhile causing maximal damage to cancer cells.

Comparative assays using WZB-27, WZB-115, as well as two knownanticancer drugs, cisplatin and taxol, were completed. In Table 4, thepercent cell death in cancer cell lines as well as normal cell lines isshown. Compounds WZB-27 and WZB-115 kill approximately the same percentof lung cancer cell line H1299 as taxol while killing significantly lessof the normal lung cell line, NL20. Comparing the breast cancer cellline MCF7, WZB-27 and WZB-115 kill somewhat fewer cells relative totaxol but more than cisplatin. Both WZB-27 and WZB-115 kill fewer of thenormal breast cell line MCF12A than taxol or cisplatin. WZB-27 killsfewer of the normal breast cell line MCF12A than taxol or cisplatinwhile WZB-115 kills no more normal cells than either cisplatin or taxol.The results shown in table 4 suggest that compounds have cytotoxicitiesin cancer cells comparable or better than cisplatin and/or taxol whilethey exhibit less cytotoxicities in non-cancerous (“normal”) cells thanthe anticancer drugs.

TABLE 4 Comparison of % cell death in cancer vs. normal cells induced bycompounds^(a,b,c) Compound H1299 NL20 MCF7 MCF12A Cisplatin 26.4 ± 3.858.4 ± 7.0 27.0 ± 1.9 69.6 ± 2.9 Taxol 45.6 ± 4.9 53.8 ± 4.2 61.4 ± 7.173.7 ± 5.6 WZB-27 52.3 ± 9.4 0 48.9 ± 5.7 21.1 ± 8.9 WZB-115 61.3 ± 3.634.1 ± 8.7 51.6 ± 8.3 66.0 ± 8.6 ^(a)This test is done using a standardMTT assay to measure viable cells after compound treatment.^(b)Concentration used in the test was the IC₅₀ for each compound.^(c)Non-compound treated cells were used as controls (0% death)

Cancer Cell Lines Overexpress GLUT1 and Compounds Inhibit More GlucoseUptake in Cancer Cells than their Non-Cancerous Cell Counterparts

To determine the possible causes for increased killing in cancer cellsthan in their non-cancerous cell counterparts, comparison studies wereconducted to determine the effect of compound treatment on glucoseuptake. It was found that compound WZB-27 produced larger reductions inglucose uptake in cancer cell line H1299 and MCF7 as compared to thereduction in non-cancerous cell lines NL20 or MCF12A (FIGS. 19A and19B). Western blot analysis revealed that these same cancer cell linesexpress significantly higher levels of GLUT1 protein than theirnon-cancerous counterparts (FIG. 19C). The larger reduction in glucoseuptake observed in cancer cell lines was correlated with higher GLUT1levels in these cells.

α-PGG Activates p53 in RKO (Colon) Cells but not in HeLa (Cervical) orMCF-7 (Breast) Cancer Cells

After it was found that α-PGG induced apoptosis in the three cancer celllines, knowledge of the mechanism of apoptosis was sought (i.e., isapoptosis related to p53 status or not). Western blot analysis usinganti-p53 antibody revealed that α-PGG led to activation of p53 in RKOcells but not in HeLa or MCF-7 cells (FIG. 6). This result suggests thatthe apoptosis induced in HeLa and MCF-7 cells was p53-independent. Thisresult is both interesting and important in that it shows that α-PGG caninduce apoptosis in certain cancer cells using a p53-independentmechanism, which should be effective in inducing apoptosis in more than50% of all human cancers in which p53 is mutated and non-functional.

This result suggests that, unlike in RKO cells, the apoptosis induced byα-PGG in HeLa cells and MCF-7 was not mediated by p53 or p53 signalingpathway. Thus, apoptosis induced by α-PGG in HeLa cells isp53-independent.

PGG and its Derivatives Inhibit Basal Glucose Transport in Human CancerCell Lines

Inhibition of basal glucose transport was speculated as a cause forcancer cell death induced by α-PGG. PGG derivatives were synthesized andtested along with α-PGG in different human cancer cell lines. Thesederivatives were found to inhibit basal glucose transport in cervical,colon, and breast cancer cell lines (FIG. 7).

Insulin had no effect on the glucose uptake, suggesting that the glucosemeasured was basal glucose transport, not insulin-mediated glucosetransport. Based on this observation, we further hypothesized that thepronounced glucose transport inhibition might be the cause of apoptosis,particularly in HeLa cells.

α-PGG and its Derived Compounds Inhibit Basal Glucose Transport in HeLaCells in a Dose-Dependent Manner

Different concentrations of α-PGG or its derived compounds, WZB-25 andWZB-27, were used to determine if the inhibition of the basal glucosetransport was dose-dependent. The experimental results indicated thatα-PGG and its derivatives inhibited the basal glucose transport in adose-dependent manner and α-PGG appears to be slightly more potent thanits derivatives in inhibiting the transport (FIG. 8). It was also foundthat 30 μM of any compound led to approximately 50% inhibition of basalglucose transport in HeLa cells (FIG. 8), suggesting that all 4compounds were about equally effective in inhibiting basal glucosetransport in HeLa cells. This result led us to conclude that we couldeither use α-PGG or substitute α-PGG with its derivatives to do theglucose transport inhibition study. We have also found that reducingglucose concentration in cell culture media also reduces cell growthrates and induces apoptosis in HeLa and MCF-7 cells in a glucoseconcentration-dependent manner (data not shown). These results clearlyshow that α-PGG and its derivatives WZB-25 and WZB-27 inhibit basalglucose transport and the inhibition is dose-independent.

HeLa cells were incubated with α-PGG or its derivatives (WZB-25 orWZB-27) at various concentrations for 20 min before ³H-labeled 2-DG wasadded to the cells for 30 min. After 30 min of 2-DG incubation, cellswere harvested, lysed, and counted for their respective glucose uptake(intracellular 2-DG counts) with a scintillation counter. The resultsshow that α-PGG starts to inhibit glucose transport at 5 μM, while otherderivatives start to inhibit the transport at about 10 μM and all threecompounds show dose-responsive inhibition profiles. Error bars in FIG. 8represent standard deviations of the measurements. Samples were done intriplicates and the experiment was repeated three times.

Unlike α-PGG (an Insulin Mimetic), α-PGG Derivatives do not Induce AktPhosphorylation

So far, it has been shown that α-PGG and its derivatives inhibited basalglucose (FIG. 7) and exhibited very similar dose-response (FIG. 8).However, these derivatives are much smaller than α-PGG in molecularweight and it is expected that these derivatives will be cleaner thanα-PGG in that they are more selective and do not have as many activitiesunrelated to the basal glucose transport inhibition. Previous datashowed that α-PGG binds and activates the insulin receptor (IR), andinduces the phosphorylation of Akt, a protein factor involved in IRsignaling. Since the derivatives are simpler in structure and smaller insize than α-PGG, it was speculated that these derivatives would notinduce Akt phosphorylation, which was confirmed by Western blot analyseson compounds-treated CHO cells that overexpress IR (FIG. 10).

Akt is a key factor in the insulin receptor signaling pathway.Therefore, α-PGG derivatives WZB-25 through WZB-27 appear to be moreselective than α-PGG in that they do not induce activities unrelated tobasal glucose transport. They should generate glucose transportinhibition and apoptosis data that are even easier to analyze andinterpret than those of α-PGG.

Basal Glucose Transport Inhibitors Induce Apoptosis and Cell Death Usinga p53-Independent Signaling Pathway

To determine if anti-cancer compounds W25 and W27 kill cancer cellsusing a p53 dependent or a p53-independent pathway, a cell killing assaywas performed in RKO and RKO E6 cell lines. The difference between thetwo cell lines is that RKO cells contain much higher levels of p53 thanRKO E6 cells. The fact that W25 and W27 killed about the same amounts ofRKO cells and RKO E6 cells in the assay (FIG. 12A) suggests that p53 inthe cancer cell lines did not play any important roles in the cellkilling. Furthermore, the PARP assay revealed that, in the W25 and W27treated H1299 cells, the intact 116 kDa PARP protein was cleaved into a89 kDa protein (FIG. 12B), indicating that the caspase 3 was activatedand caspase 3 apoptosis pathway is active in the compound treated lungcancer cells. These results suggest that the compounds kill cancer cellsusing a p53-independent and caspase 3-dependent apoptosis pathway.

Although it was found that there was no significant difference betweenthe viability of RKO and RKO-E6 cells after they were treated withcompound WZB-25 or WZB-27 (FIG. 18A), a significant difference inviability between the two cell lines was observed when they were treatedby α-PGG (FIGS. 18A and 18B), suggesting that the apoptosis induced byWZB-25 or WZB-27 is p53-independent, since p53 levels in the two celllines did not affect cell viability. In contrast, the apoptosis inducedby α-PGG is p53-dependent since its treatment led to very different cellviability in the same two cell lines (FIG. 18B). This finding isimportant because it is known that more 50% of all human cancers harborp53 mutations. These inhibitors should be able to exert their anticancereffects on all human cancers regardless their p53 status.

Furthermore, a Western blot analysis revealed that, in the WZB-27 andWZB-115 treated A549 cells, the intact 116 kDa PARP protein was cleavedinto a 89 kDa protein (FIG. 18C), indicating that the caspase 3 wasactivated and caspase 3 apoptosis pathway is active in the compoundtreated lung cancer cells. More interestingly, cells growing in cellculture media containing 5% of normal glucose concentration (1.25 mM vs.25 mM normal) also demonstrated the cleaved 89 kDa band (FIG. 18C),indicating the glucose withdrawal resulted in the same PARP cleavage.These results suggest that the compounds WZB-27 and WZB-115 induceapoptosis in these cancer cell lines using a p53-independent and caspase3-dependent apoptosis pathway while α-PGG does this in a differentp53-dependent mechanism. It also reveals that cell treatments by thecompound inhibitors produced the same PARP cleavage as glucosewithdrawal, providing initial experimental evidence that compoundtreatment mimics the effects produced by glucose withdrawal. This isimportant since it suggests that the methods of inhibition of basalglucose transport by inhibitors and glucose withdrawal by reducingglucose concentration in cell growth media may be interchangeable whenin producing certain biological effects in cells.

Mechanism of Compounds in Cancer Cell Killing

Potential mechanisms for cancer cell killing are graphically presentedin FIG. 13. According to the hypothesis, extracellular glucose is takenup by cancer cells through glucose transporter 1 (GLUT1). Compound α-PGGand its derivatives inhibit basal glucose transport by inhibiting GLUT(most likely to be GLUT1). The inhibition of basal glucose transportresults in reduction of intracellular glucose concentration and anincrease of intracellular free Zn²⁺; these changes, through an unknownmechanism(s), induces ER stress and eventually apoptosis (FIG. 11).

Glucose Transport Inhibitors Induced Mild and Temporary Hyperglycemia inFasting Mice

Inhibitors to basal glucose transport are likely to increase bloodglucose level when used in animals because the movement of blood glucoseinto target cells, primarily muscle and fat cells, is partially blockedby the inhibitors. To find out the intensity and duration of the inducedhyperglycemia and other potential side effects in animals, we performedanimal studies by injecting two lead compounds WZB-27 and WZB-115separately and intraperitoneally (IP) into fasting mice and observingblood glucose changes, movement, and behaviors of the compound injectedanimals. As expected, the compound-injected mice showed mild andtemporary hyperglycemia compared to the vehicle-injected group(PBS+DMSO) and the hyperglycemia went away approximately 3 hrs after thecompound injection (FIG. 20). Both compounds WZB-27 and WZB-115 showedvery similar blood glucose profiles (FIG. 20). At IC₅₀ (10 mg/kg forWZB-27 and 1.5 mg/kg for WZB-115), both compounds induced mild andtemporary hyperglycemia. Interestingly, at a concentration of 3×IC₅₀,neither compound induced higher hyperglycemia. Instead, injection of3×IC₅₀ (30 mg/kg for WZB-27 and 5 mg/kg for WZB-115) resulted in bloodglucose levels very similar to that of the vehicle injected group (FIG.20). This experiment was repeated once and similar results wereobtained. Although the mechanism for normoglycemia at higher compoundconcentrations was currently unclear, it was concluded that thesecompounds do not produce severe hyperglycemia in mice. No othernoticeable changes in animal movement, activity, and behavior wereobserved in this experiment.

To further address the concerns of animal side effects, a longer-termmultiple compound injections at higher concentration were performed.Animals were injected once a day at a concentration of 6×IC₅₀ or 10×IC₅₀for one week. Similar to the single day experiment, no noticeable sideeffects were observed except mild and temporary hyperglycemia in bothgroups and some weight loss for the 6×IC₅₀ group (≤10% of total bodyweight). There was no significant difference between blood glucose ofthe compound treated group at the end of the one week study and that ofthe vehicle treated group. These results strongly suggest thatinhibition of basal glucose transport may not be very toxic and arerelatively safe to mice. The anticancer animal studies disclosed hereincan be carried out to determine the in vivo anticancer efficacy of thecompounds.

Comparison of Intracellular Glucose Levels in Glucose DeprivationInduced by Either Glucose Removal or by Inhibition of Basal GlucoseTransport

Glucose deprivation experiments, in which glucose in the cell culturemedia is partially removed, have been frequently done. However, theintracellular glucose level changes have not been measured often duringor after the deprivation. In order to compare the effects of glucosewithdrawal and basal glucose transport inhibition on intracellularglucose levels, a comparison study will be conducted.

H1299 and MCF7 will be incubated in 24-well plates in their regularmedia with a glucose concentration of 25 mM overnight and intracellularglucose concentrations of the cells should be in equilibrium with theextracellular glucose concentration. The glucose concentration in cellgrowth media of some wells is reduced by mixing regularglucose-containing medium with glucose-free medium at different ratiosto achieve the following final glucose concentrations 25 mM, 10 mM, 2.5mM, 1 mM, 0.25 mM (1% of the original concentration). The media will besupplemented with of ³H-deoxyglucose at 1/50 of the cold glucoseconcentration and then added to cells. The cells will be incubated for30, 60, and 120 min, and the intracellular ³H-DG will be measured byscintillation counter after media removal and cell lysis as in astandard glucose uptake assay. These samples treated by glucose removalcan be considered as positive controls of the experiment. Forcomparison, cancer cells in wells with regular growth mediumsupplemented with ³H-DG will be treated with different concentrations ofknown glucose transport inhibitors fasentin, apigenin, or anti-GLUT1antibody and then have their intracellular ³H-DG levels measured at thesame times post treatment as the glucose removal samples. Dose responseand time response curves can be generated from these data and thencompared. These curves will reveal if the glucose removal and inhibitionof basal glucose transport lead to the same or different intracellularglucose concentrations. Our in house glucose transport inhibitor WZB-27and WZB-115 will also be used in the experiment and will be compared tothose samples treated by fasentin, apigenin, or anti-GLUT1 antibody.Cells not treated by either glucose withdrawal or compound (but withsame amount of radioactive ³H-DG) will serve as untreated baseline(negative) controls.

Measurement of GLUT1 Protein and mRNA Levels During Glucose Removal orInhibition of Basal Glucose Transport

It is known that GLUT1 protein and mRNA are down-regulated when glucoseis withdrawn. However, it is not known if the inhibition of basalglucose transport induced by fasentin or our inhibitor compounds alsoresults in GLUT1 down-regulation. To answer this question, cancer cellsH1299 and MCF7 cells are treated with (1) glucose withdrawal (removalfrom cell culture media) at multiple glucose concentrations, and (2)inhibition of basal glucose transport by the compounds (fasentin,apigenin, anti-GLUT1 antibody, and compounds WZB-27 and WZB-115) atmultiple compound concentrations. After treatments for 1, 2, 4, 8, 12,24 or 48 hrs, the cells are harvested and total cellular proteins areisolated. A western blot analysis is carried out using anti-GLUT1antibody (from Santa Cruz) to compare GLUT1 protein levels in thesamples of two different treatments. Each GLUT1 protein band will bequantified using densitometry and then normalized with its own β-actinprotein loading control, and compared to GLUT1 level in the untreatedcontrol samples. The comparison between treated samples and untreatedsamples will tell us whether the treatments lead to down-regulation ofGLUT1 protein. The comparison between samples of glucose removal andsamples treated by compounds will show whether these two treatmentsresult in differences in GLUT1, while the comparison betweenfasentin/apigenin/anti-GLUT1 antibody treated samples and those treatedby WZB-27/WZB-115 will reveal the similarity and difference among thesecompounds.

In the same experiment, total RNA will also be isolated. Commerciallyavailable primers unique to GLUT1 will be purchased and used inreal-time PCR to quantify GLUT1 mRNA levels in each treatmentconditions. GAPDH and/or j3-actin mRNA will be used as internal RNAcontrol. Comparisons will be made among these treatments with untreatedsamples (control) and between glucose removal and inhibition of basalglucose transport by compounds. These comparisons will show whetherGLUT1 is also down-regulated at the mRNA level and whether these twotreatments lead to different GLUT1 mRNA expression results.

Measurement of Glycolysis Rates Under Different Experimental Conditions

H1299 lung cancer and MCF7 breast cancer cells will be treated with orwithout glucose removal or inhibitor of basal glucose transport.Glycolysis rates of each treatment condition will be measured bymonitoring the conversion of 5-³H-glucose to ³H₂O, as describedpreviously. Briefly, 10⁶ of H1299 and MCF7 cells are washed once in PBSprior to re-suspension in 1 ml of Krebs buffer and incubation for 30 minat 37° C. Cells are then pelleted, re-suspended in 0.5 ml of Krebsbuffer containing glucose (10 mM, if not specified), and spiked with 10μCi of 5-³H-glucose. Following incubation for 1 h at 37° C., triplicate50 μl aliquots are transferred to uncapped PCR tubes containing 50 l of0.2 N HCl (for stopping the reaction), and a tube is transferred to ascintillation vial containing 0.5 ml of H₂O such that the water in thevial and the contents of the PCR tube are not allowed to mix. The vialswill be sealed, and diffusion is allowed to occur for a minimum of 24 h(to reach equilibrium). The amounts of diffused and undiffused ³H aredetermined by scintillation counting. Appropriate ³H-glucose-only (nocell) and ³H₂O-only controls will be included in the assay, enabling thecalculation of ³H₂O in each sample and thus the rate of glycolysis.Glucose utilization rate will be calculated as ³H H₂O formed from³H-glucose, expressed in the term of pmol of glucose utilized/10⁶ cancercells from the formula:

${{Glucose}\mspace{14mu} {utilized}\mspace{14mu} ({pmol})} = \frac{\left\lbrack {\,^{3}H} \right\rbrack \mspace{14mu} {water}\mspace{14mu} {formed}\mspace{14mu} \left( {d.p.m.} \right)}{\begin{matrix}{{{sp}.\mspace{14mu} {radioactivity}}\mspace{14mu} {{of}\mspace{14mu}\left\lbrack {5\text{-}^{3}H} \right\rbrack}} \\{{glucose}\mspace{14mu} \left( {{d.p.m.\text{/}}{pmol}} \right)}\end{matrix}}$

These measurements will enable us to determine how glucose withdrawaland compound inhibition affect glycolysis rates in cancer cells.Untreated cancer cells will be used as baseline controls. Non-cancerouscell counterparts NL-20 (normal lung) cells and MCF12A (normal breast)cells will also be used as controls for comparison.

Glycolytic Enzymes Alteration During Glucose Withdrawal or Basal GlucoseTransport Inhibition

It has been shown that glucose withdrawal resulted in down-regulation ofglycolytic enzymes such as hexokinase and pyruvate kinase (PK). PKM2 hasbeen found to be very important for tumorigenesis and exclusivelyexpressed in cancer or proliferating cells. It is still unclear ifinhibition of basal glucose transport by fasentin or our compoundinhibitors also leads to similar results. To answer this question, H1299and MCF7 cancer cells growing in 24-well cell culture plates will betreated with or without anti-GLUT1 antibody, fasentin and our inhibitorcompounds WZB-27 and WZB-115 at their respective IC₅₀ and IC₇₀. After 24and 48 hr treatment, cells will be harvested and total protein isisolated. Western blot analyses will be performed to compare the levelsof PKM2 in the treated and untreated samples using PK antibodies (CellSignaling). This study will enable us to know exactly what happens toPKM2 when cancer cells are treated with compound inhibitors. Same cancercells treated with or without glucose withdrawal will be included in thestudy for comparison.

The activity of hexokinase, the first enzyme in glycolysis, will also bestudied by a similar method described for PK. Antibodies againsthexokinase are commercially available. In addition, the enzymaticactivity of hexokinase will also be measured and used as an indicationof changes in glycolysis as hexokinase is the enzyme catalyzing thefirst rate-limiting step of glycolysis. Hexokinase activity will bemeasured using a published protocol. Briefly, the activity will bedetermined spectrophotometrically at 30° C. by coupling the formation ofglucose 6-phosphate with its removal via glucose-6-phosphatedehydrogenase, during which the absorbance of NADPH at 340 nm changes.Activity is expressed in mUs, 1 mU defined as the formation of 1 nmolNADPH/min. Enzyme was dissolved in Tris.MgCl2 buffer, pH 8.0 to obtain arate of 0.02-0.04 AA/min. The assay medium contained 0.05M Tris.MgCl₂buffer, pH 8.0, 15 mM MgCl₂, 16.5 mM ATP, 6.8 mM NAD, 0.67 mM glucose,and 1.2 units/ml glucose-6-phosphate dehydrogenase. Incubate in thespectrophotometer at 30° C. for 6-8 minutes to achieve temperatureequilibration and establish blank rate, if any. At zero time, add 0.1 mlof diluted hexokinase solution and mix thoroughly. Record increase inabsorbance at 340 nm for 3-4 minutes. Determine AA/min from initiallinear portion of curve.

Calculation will be performed using the formula shown below:

${{Units}\text{/}{mg}\mspace{14mu} {protein}} = \frac{{\Delta A}_{340}\text{/}\min}{6.22 \times {mg}\mspace{14mu} {enzyme}\text{/}{ml}\mspace{14mu} {reaction}\mspace{14mu} {mixture}}$

Studies Related to Protein Factors Signal Transduction During GlucoseDeprivation Induced by Inhibition of Basal Glucose Transport

The activation of Akt was found to increase the rate of glycolysispartially due to its ability to promote the expression of glycolyticenzymes through HIFα. This was speculated as a major factor contributingto the highly glycolytic nature of cancer cells. It would be alsointeresting to find out how changes in glucose transport and glycolysisaffect expression of Akt.

In this experiment, cancer cell H1299 and MCF7 will be treated with orwithout anti-GLUT1 antibody, fasentin, WZB-27, or WZB-115 at theirrespective IC₅₀. Differentially treated cells will be harvested 1, 2, 4,8, 24 hrs after the treatment. Total proteins from each sample will beisolated and analyzed by western blots using antibodies specificallyagainst Akt. Akt has multiple phosphorylation sites and differentantibodies will be used to distinguish Akt phosphorylated at differentsites. Treated samples will be compared to untreated samples and sampleswith different treatments will also be compared. Changes in intensity oftotal Akt protein as well as changes in different phosphorylated formsof Akt will specify how inhibition of basal glucose transport affectsexpression and phosphorylation of Akt. Another protein factor involvedcell growth signaling pathway, AMPK, which has been found to affectglycolysis, will also be studied the same way as Akt.

Inhibitors to Glucose Transport Sensitize and Synergize with AnticancerDrugs in Cancer Cell Killing

The inhibitors disclosed herein target basal glucose transport whileother anticancer drugs target pathways or processes not directly relatedto glucose transport. As a result, it was speculated that glucosetransport inhibitors could potentiate or synergize with other anticancerdrugs in their cancer killing activity when used together. This would beconsistent with the recent finding that anti-GLUT1 antibody sensitizesand enhances the anticancer activity of anticancer drugs and ourinhibitors should do the same. This has been shown to be the case in acompound study in H1299 or A549 cells lung cancer cells, which weretreated with WZB-115 or (WZB-115+cisplatin) or (WZB-115+taxol) (FIG.21). Addition of WZB-115 to either cisplatin or taxol led tosignificantly more cancer cell killing than that induced by drugs alone.Similar results have been obtained for WZB-27 as well (FIG. 21).

This result suggests that glucose inhibitors such as WZB-115 or WZB-27could significantly enhance the cytotoxic activity of anticancer drugs.This is accomplished by the compound's independent anticancer activityor the sensitizing activity of the compound to anticancer drugs or both.Further mechanistic study should be able to determine the realmechanism(s). This result, similar to what others found in their glucosetransport inhibitor studies, strongly suggests that these compounds canbe used alone to inhibit cancer growth or used together with otheranticancer drugs to further increase the anticancer efficacy of thedrugs. This also suggests that we may not have to use these inhibitorsat very high concentrations as long as they are co-administered alongwith other anticancer drugs.

Inhibitors are More Potent than Known Inhibitor Fasentin in Both GlucoseUptake Inhibition and in Reducing Cancer Viability

Fasentin is a published inhibitor of basal glucose transport and knownGLUT1 inhibitor. A comparison of reported inhibitors with fasentin wascompleted to determine if the compounds of interest exhibited similarbiological activities to fasentin. It has been found that, similar tofasentin, our inhibitors induce reduction of glucose uptake. Somecompounds such as WZB-115, 131, and 133 demonstrated stronger inhibitionthan fasentin in cancer cells (FIG. 22A). In addition, addition of thesecompounds (WZB-131 and 133) to different cancer cell lines resulted inmore cancer cell death than fasentin at the same concentration (FIG.22B).

These results demonstrated that (a) these compounds exhibit biologicalactivities similar to fasentin, they are true inhibitors of basalglucose transport like fasentin, (b) they are more potent than fasentinin both activities tested. All these results indicate that thesecompounds are fasentin-like and are inhibitors of glucose transport butthey possess more potent anticancer activities. As a result, they can beused in studies of glucose transport, glycolysis, and apoptosis ofcancer cells as potentially superior inhibitors than fasentin.

Anticancer Activity Screening of Compound WZB-115 in 59 Cancer CellLines Done by NCI

In order to further evaluate the lead compound WZB-115, the compound wassent to the National Cancer Institute (NCI) for screening its anticanceractivities in a total of 59 cancer cell lines (FIG. 23). The screeningresults indicate that, (a) among 59 cancer cell lines and at 10 μM, thecompound reduced the growth rates of 51 cell lines by more than 10%(<90% of the growth rates of the controls). (b) The compound reduced thegrowth rates of 21 cancer cell lines by more than 50% (or 35.6% of the59 lines tested, FIG. 23). This result suggests that, in these 21 lines,the IC₅₀ of WZB-115 is lower than 10 μM. (c) The compound showsanticancer activities in all cancer types although it may be moreeffective in certain cancer types than others. (d) Large variations inactivities are observed among cancer cell lines both within a singlecancer type or among different cancer types. As a result, this compoundis less likely to be very cytotoxic to normal cells than those compoundsthat are equally cytotoxic to all cancer cell lines. This is alsoconsistent to our observation in animal injection tests (FIG. 20).Because of these promising results, the compound is recommended by NCIfor a second round of screening using 5 different concentrations todetermine its IC₅₀s in all these different cancer cell lines.

In summary, the preliminary results indicate that these compounds areinhibitors of basal glucose transport in all cancer cell lines tested(Table 3 and FIG. 19). The compound treatment led to apoptosis that isp53-independent and caspase 3-dependent (FIG. 18). The compoundtreatment caused significantly more cell death in cancer cells than intheir normal cell counterparts (Table 4). Their preferential cancer cellkilling also indicates that these cancer cell lines are more sensitiveto the compound treatment than their normal cell counterparts, stronglysuggesting these compounds are significantly more toxic to cancer cellsto normal cells. They only cause mild and temporary hyperglycemia inanimals (FIG. 20) without other noticeable side effects. Furthermore,they potentiate and synergize with existing anticancer drugs (FIG. 21).The addition of the compounds led to phenotypic changes in cancer cellsas induced by glucose deprivation (FIG. 18C). These compounds form anovel group of molecular tool and anticancer agents since they inhibit anew target, basal glucose transport. Fasentin and these compounds can beused for studying how glucose deprivation affects glycolysis and howchanges in glycolysis affect other changes such as apoptosis.

Initial Animal Efficacy and Safety Study

The ability of compounds WZB-27 and WZB-115 to inhibit/reverse tumorgrowth in nude mice, as well as the clinical safety of the compoundswill be determined. Compounds will be use to treat nude mice (JacksonLabs) with cancer grown from H1299 (lung cancer) and MCF-7 (breastcancer) cells. Five millions of cancer cells will be injectedsubcutaneously into the flank of each of 30 nude mice. The tumorcell-injected mice will be randomly split into three groups: ten forcompound treatment, ten for drug (e.g. WZB-117) treatment (positivecontrols) and another ten receive vehicle (solvent) treatment. Aftertumors become palpable and visible (˜5-7 days), the compound treatmentwill begin. A molar concentration of IC₅₀ will be chosen for eachcompound for the treatment. Compounds will be dissolved in DMSO or othercompatible solvent right before injection. The solvent that dissolve thecompounds will be used in the solvent treatment group. The animal studydesign is shown in Table 5.

TABLE 5 Design of initial animal study (same study in both H1299 andMCF-7 tumor mice) Treatment Group/group function N= Treatment durationParameters measured 1. Negative control 10 Solvent, once a day 4-5 wksTumor size, blood glucose, body weight 2. Fasentin, positive 10 Fasentinat IC₅₀*, as above 4-5 wks Same as above control 3. WZB-27 10 WZB-27 atIC₅₀, as above 4-5 wks Same as above 4. WZB-115 10 WZB-115 at IC₅₀, asabove 4-5 wks Same as above *IC₅₀ is determined from cancer cellviability study. IC₅₀ is different for each cancer cell line

The IP injection of compounds will be performed once every day for 4-5weeks depending upon tumor growth rates. Tumor sizes will be measuredwith calipers twice a week and recorded as LW²/2=volume in mm³(L=length, W=width) and compared to those of tumors on solvent injectedcontrol mice. Body weight of the mice is measured once a week. Bodyweight is an indication of the health status of the treated mice. Inorder to determine how compound treatment affects blood glucose levels,blood glucose will also be measured immediately prior to the compoundinjection and 1, 2, and 4 hr after the injection and the blood glucoselevels will be compared to those of the solvent injected mice. Once theglucose levels are measured, a decision will be made on if the bloodglucose monitoring should be continued or can be terminated. Thecompound treatment lasts 3-5 weeks until the tumors grown in the solventtreated mice become large (>5% but <10% of the body weight, or ≥20 mm inthe largest length measurement). The animal study will be carried outand terminated in accordance to the rules and regulations of NIH and ofour university IACUC. Tumor-bearing mice will be euthanized at the endof the study, according to the related rules by NIH and DOA. The averagesize of the tumors in the treated groups will be compared to theuntreated control group to show treatment efficacy and statisticaldifferences. The better of the two compounds, based on combinedconsideration of anticancer efficacy and toxicity to mice (primarily itseffect on blood glucose levels and body weight changes and/or otherunexpected side effects), will be chosen for the dose response animalstudy described below.

Determination of Dose Response of Compound Treatment in Animals

Although anticancer efficacy may be shown in the initial animal study,the dose used in the study is definitely not optimal for each compound.In order to further determine the better dose, at which anticancerefficacy is maximized but the side effects are still tolerable, a doseresponse animal study will be conducted (Table 6).

TABLE 6 Determination of compound dose response Treatment Group/groupfunction N Treatment duration Parameter measured 1. Negative control 10Solvent 4-5 wks Tumor size, blood glucose levels, body weight 2.Fasentin low dose 10 Fasentin at IC₅₀ 4-5 wks Same as above 3. Fasentinhigh dose 10 Fasentin at 3x IC₅₀ 4-5 wks Same as above 4. WZB-27 lowdose 10 WZB-27 at IC₅₀ 4-5 wks Same as above 5. WZB-27 high dose 10WZB-27 at 3x IC₅₀ 4-5 wks Same as above 6. WZB-115 low dose 10 WZB-115at IC₅₀ 4-5 wks Same as above 7. WZB-115 high dose 10 WZB-115 at 3x IC₅₀4-5 wks Same as above

For compounds Fasentin, WZB-27 and WZB-115, two doses will be tried:IC₅₀ and 3×IC₅₀. After this experiment, The dose response for thesecompounds will be known. We will also know which compound performs thebest in terms of reduction of tumor size and side effects, and if the3×IC₅₀ dose can be well tolerated by nude mice or not. This study willbe done using both H1299 and MCF-7 cancer cell models. From the resultsof this experiment, one of the two compounds, WZB-27 or WZB-115designated as compound to be determined (Compound_(tbd)), and its betterdose, will be selected for the next round of animal study.

Does Inhibitor of Basal Glucose Transport Potentiate and Synergize withCancer Drugs in the Anticancer Activity

Synergistic and potentiating effects were found between fasentin andanticancer drugs (Ref). In our preliminary studies, the similar effectswere also observed (FIG. 21). However, it is unclear if such synergisticeffects can also be found in animals. To that end, a large animal studywill be conducted (Table 7).

TABLE 7 Design of animal study for determination of synergy betweeninhibitors and cancer drugs Treatment Group/group function N Treatmentduration Parameter measured 1. Negative Control 10 Solvent 4-5 wks Tumorsize, blood glucose levels, body weight 2. Fasentin 10 Fasentin atC_(tbd)* 4-5 wks Same as above 3. Compound 10 Compound_(tbd)** atC_(tbd) 4-5 wks Same as above 4. Cisplatin 10 Cisplatin at IC₇₀ 4-5 wksSame as above 5. Taxol 10 Taxol at IC₇₀ 4-5 wks Same as above 6.Fasentin + cisplatin 10 Fasentin at C_(tbd) + 4-5 wks Same as aboveCisplatin at IC₇₀ 7. Compound + cisplatin 10 Compound at C_(tbd) + 4-5wks Same as above Cisplatin at IC₇₀ 8. Fasentin + taxol 10 Fasentin atC_(tbd) + 4-5 wks Same as above taxol at IC₇₀ 9. Compound + taxol 10Compound at C_(tbd) + 4-5 wks Same as above taxol at IC₇₀ *C_(tbd) =concentration to be determined as described herein; **Compound_(tbd) =compound to be determined as described herein.

How Compound Inhibitor Treatment Affects Levels of Proteins/EnzymesInvolved in Glycolysis, Cell Growth Signal Transduction, and Apoptosis

To better understand how inhibitors of basal glucose transport inhibittumor growth in vivo, tumors treated with or without compound inhibitorswill be removed from tumor mice euthanized at the end of animal studiesdescribed above and are immediately frozen by liquid nitrogen for lateanalysis. For tumor analysis, proteins will be extracted from removedtumors and quantified. Protein samples will then be subjected to PAGEfollowed by western blotting analysis using antibodies specificallyagainst p53, Akt, PKM2, hexokinase, and caspases. The intensities ofthese proteins from compound treated samples will be compared to thoseof tumor samples that are treated by solvent (vehicle). Protein β-actinand/or GAPDH will be used as protein loading controls for normalizingprotein bands. These western blots will enable us to gain the proteinexpression changes in the compound treated tumors, which can potentiallyfacilitate the final elucidation of anticancer mechanism(s) of thesecompound inhibitors in vivo.

Synthesis of Generation 2 Compounds

Disclosed herein is the design and synthesis of a second generation ofbasal glucose transporters based upon alteration of the linkage betweenthe parent aromatic ring and the phenolic aromatic substituents.Previously, a small library of polyphenolic esters were synthesized andevaluated. The generation 1 compounds were shown to inhibit basalglucose transport in H1299 lung cancer cells, and also inhibited cancercell growth in H1299 cells. WZB-115 was selected as the lead compoundfrom this library. Unfortunately, WZB-115 failed long-term stabilityassays in animal models. The degradation rate of WZB-115 and WZB-117 inhuman serum was established, both compounds degraded completely after 48hours. Thus, more stable analogs need to be designed and synthesized.

Disclosed herein are the structures of novel glucose transportinhibitors WZB-115, WZB-117, and WZB-173 (FIG. 37). Compound WZB-117 isan analog of WZB-115 while WZB-173 is an ether-bond analog. CompoundsWZB-117 and WBZ-173 (FIG. 37) are derived from compounds WZB-115, whichis a polyphenolic model compound used in our generation 1 cancerstudies. WZB-115 was derived from a natural anticancer and antidiabeticcompound called penta-galloyl-glucose (PGG). WZB-117 and WZB-173 arevery similar structurally to WZB-115 but are both structurallysimplified and functionally optimized compared to WZB-115. As a result,WZB-117 and WZB-173 are more potent in their anticancer activities thanWZB-115 and are also structurally more stable than WZB-115 in solutionand cell culture media.

Synthesis of Multi-Phenolic Ether Derivatives

3-(Methoxymethoxy)benzyl chloride, the precursor for predesignedpolyphenolic derivatives, was synthesized in high yield over four steps.Commercially available 3-hydroxybenzoic acid was treated with sulfuricacid in methanol to elicit a Fischer esterification. Protection of thephenol was accomplished by treatment of the phenolate with MOMC1.Reduction of the ester followed by a modified Appel reaction affordedthe desired compound in greater than 65% over four steps (Scheme 2).

The synthesis of the polyphenolic ethers, amines, and amides wereaccomplished via S_(N)2-type reactions or nucleophilic acyl substitutionreactions (Scheme 3 and 4). Synthesis of the second generation basalglucose transport inhibitors could be accomplished by one who is skilledin the art without further description or disclosure of furtherexperimental detail.

Compd # X¹ (OH)n X² (OR)n Yield^(a) WZB-131 OH 1,2-(OH)₂ 3-OH—C₆H₄CH₂O1,2-(3-OH—C₆H₄CH₂O)2 54 WZB-132 OMe 1,2-(OH)₂ OMe 1,2-(3-OH—C₆H₄CH₂O)₂70 WZB-133 Cl 3,4-(OH)₂ Cl 3,4-(3-OH—C₆H₄CH₂O)₂ 65 WZB-134 F 1,2-(OH)₂ F1,2-(3-OH—C₆H₄CH₂O)₂ 46 WZB-137 Cl 1,3-(OH)₂ Cl 1,3-(3-OH—C₆H₄CH₂O)₂ 57WZB-141 Cl 2,4-(OH)₂ Cl 2,4-(3-OH—C₆H₄CH₂O)₂ 72 ^(a)yield is based ontwo steps

Compd # X R² a Y Yield WZB-125 Cl H H₂, Pd/C CO 83 WZB-138 Cl H 1) H₂,Pd/C; 2) LAH CH₂ 75 WZB-142 Cl Me 1) Mel, NaH; 2) H₂, Pd/C CO 71 WZB-145Cl Me 1) Mel, NaH; 2) H₂, Pd/C; 3) LAH CH₂ 66 WZB-124 F H H₂, Pd/C CO 86WZB-139 F H 1) H₂, Pd/C; 2) LAH CH₂ 78 WZB-143 F Me 1) Mel, NaH; 2) H₂,Pd/C CO 70 WZB-144 F Me 1) Mel, NaH; 2) H₂, Pd/C; 3) LAH CH₂ 65 Theseparate yield is based on the step(s) shown in reaction conditionsabove

Evaluation of Generation 2 Compounds

Compounds WZB-134, WZB-141 and WZB-144 inhibited basal glucose transportin H1299 cells by 92.5±2.2%, 96.5±0.5%, and 78.2±1.4%, respectively(Table 12), as measured by a standard glucose uptake assay compared tonon-compound treated cells controls (considered as 0% inhibition).Tested in an MTT cell proliferation assay in H1299 cells, theirinhibitory activities on cancer cell growth were found to be 10.6±2.0%,39.5±1.8%, and 14.5±7.6%, respectively (non-compound treated cellcontrols were considered as 0% inhibition).

TABLE 12 Polyphenolic ethers, amine, and amide induced inhibitoryactivities in basal glucose transport and cell growth in H1299 lungcancer cells Glucose transport Cell growth Compound # inhibition^(a)(%)inhibition^(b)(%) WZB-124  89.7 ± 2.5^(b)  5.7 ± 2.1 WZB-125 83.7 ± 0.7 5.9 ± 1.4 WZB-131 95.0 ± 1.6 12.9 ± 2.6 WZB-132 60.0 ± 7.7  6.0 ± 2.2WZB-133 91.2 ± 0.8 14.3 ± 2.7 WZB-134 92.6 ± 2.2 10.6 ± 2.0 WZB-137 86.2± 1.6 — WZB-138 52.2 ± 9.7 — WZB-139 39.2 ± 0.8 — WZB-141 96.5 ± 0.539.5 ± 1.8 WZB-142 82.7 ± 6.8 14.2 ± 9.0 WZB-143 85.7 ± 3.6 31.1 ± 7.9WZB-144 78.2 ± 1.4 14.5 ± 7.6 WZB-145 73.2 ± 3.8 38.2 ± 6.6^(a)Untreated cells served as negative controls (0% inhibition).^(b)Data were presented as a mean ± standard deviation.

Antitumor Study. Anticancer Activity of WZB-117 Against Human LungCancer A549 Grafted on Nude Mice

This animal tumor study indicated that, by daily injection of WZB-117,the tumor size of the compound treated tumors were on averageapproximately 75% smaller than that of the mock treated group althoughthe variation of the tumor sizes were quite large (FIG. 30A). Thisresult was qualitatively similar to that of a tumor study usingantiglycolytics. Noteworthy, two of the ten compound treated tumorsdisappeared during the treatment and they never grew back even at theend of the study (FIG. 30B). Body weight measurement and analysisrevealed that the mice treated with WZB-117 lost 2-3 grams of weightcompared to the mock-treated mice with most of the weight loss in thefat tissue (Table 8). Blood counts and analysis showed that some keyblood cells such as lymphocytes and platelets were significantly changedin the compound treated mice, but they were still in the normal ranges(Table 9). All these results indicate that the treatment of WZB-117 waseffective in reducing tumor sizes and the treatment was relatively welltolerated by the mice. One of the concerns for using basal glucosetransport inhibitors is that the inhibitor may cause hyperglycemia inthe injected patient. It has been found that the injection of WZB-117produced mild and temporary hyperglycemia that disappeared within aperiod of 2 hours after the compound administration, and it did notresult in permanent hyperglycemia.

Anticancer Mechanism Study. Anticancer Compound WZB-117 Inhibition ofGlut1

To demonstrate that inhibitors of glucose transport induce ER stress, 30μM of inhibitor WZB-117 was used to treat A549 cells (with glucosedeprivation being the control). The Western blot of the proteinsisolated from the treated cells shows that glucose regulated protein-78(GRP78, also called BiP), one of the key ER stress markers, wassignificantly upregulated at 48 and 72 hrs after the inhibitor treatment(FIG. 32), indicating that the inhibitor indeed induces ER stress incancer cells. Glucose concentration in regular cell culture medium was25 mM, and 2.5 mM (10% of the regular concentration) was used as theglucose deprivation control condition. The inhibitor also led toqualitatively similar results in BiP upregulation as glucose deprivation(FIG. 32).

Glucose deprivation induces upregulation of ER stress protein BiP. Lungcancer A459 cells were treated by either glucose deprivation or byinhibitor 117 for various times and then proteins of the cells wereanalyzed by using anti-BiP antibody. β-actin serves as a protein control(FIG. 32A).

Previously, it was found that our anti-glucose transport compoundsinhibited glucose transport in all the cancer cell lines tested. It wasspeculated that the target of these inhibitors is Glut1 since Glut1 isresponsible for basal glucose transport in almost all cell types. Inorder to test this hypothesis, RBC was chosen as a cell model to studybecause RBC has been known to express only Glut1, not any other glucosetransporters. The glucose uptake assays revealed that WZB-117 indeedinhibited the glucose transport in RBC (FIG. 31A), supporting the notionthat WZB-117 inhibits glucose transport by inhibiting Glut1. To furthereliminate other possibilities, the glucose uptake assays were repeatedin RBC-derived vesicles, in which all the intracellular proteins andenzymes were removed and only membrane-bound and tightly associatedproteins left. The assay result showed that WZB-117 continued to inhibitglucose transport in these vesicles, indicating that intracellularproteins are not needed for the inhibition and providing strong evidencethat Glut1 is the target of the inhibition (FIG. 31C-E).

In order to determine the protein target of the inhibition of basalglucose transport by our compound, human red blood cells (RBC) wereused. The selection of RBC was based on (1) Glut1 was hypothesized asthe most probable target and RBC express Glut1 as their only glucosetransporter, (2) RBC are an established model for and have beenfrequently used in studying glucose transport.

It was found that, in addition its inhibition of basal glucose transportin all the cancer cell lines, our compound inhibits glucose transport inRBC (FIG. 31), indicative that the compound acts on Glut1 for theinhibition and supportive that Glut1 is the target of the inhibition.

To further the target identification, vesicles were prepared from theruptured RBC called ghosts. These small sealed vesicles were formed fromplasma membrane of RBC under different salt conditions and theydemonstrated two distinct orientations: inside out or right side out.The right side out vesicles (ROV) exhibit the same membrane orientationas RBC while inside out vesicles (IOV) show opposite membraneorientation as the membrane of RBC. However, since Glut1 is a glucoseuniporter and can transport glucose in both directions, Glut1 located oneither IOV or ROV should be able to transport glucose down the glucosegradient. As expected, the glucose uptake assays showed that ourcompound could inhibit Glut1-mediated glucose transport in both IOV andROV (FIG. 31C-E), providing strong supporting evidence for ourhypothesis that Glut1 is the target of the action of the inhibition byour compound. The reason that the compound showed more inhibition in IOVthan in ROV was due to the presence of Mg⁺⁺ in the ROV preparation. Mg⁺⁺is not previously known for its interference with Glut1 function.

The observation that the compound worked on both ROV and IOV alsosuggests that the compound may interact with the excellular portion ofGlut1. The extracellular portion of Glut1 is located intracelluarly(intravesicularlly) in IOV. The compound is likely to cross the vesiclemembrane and interact with the intravesicular part of Glut1, inhibitingthe glucose transport down the gradient. This is consistent with thechemical properties of the compound, which indicates the hydrophobicityof the compound is likely to allow the compound to cross the vesiclemembrane and function intravesicularlly in artificial vesicles orintracellularly in intact cells.

Generation 2 Compounds Induce Cell Death Preferentially in Cancer CellsOpposed to their Normal Counterparts

Cancer cells depend on glucose as their energy source and basal glucosetransport inhibition has been proposed as an anti-cancer strategy. Inorder for these compounds to be effective anti-cancer agents, they mustbe able to kill more cancer cells than normal cells. Compound WZB 117kills significantly more cancer cells than non-cancerous cells. A549lung cancer and MCF7 breast cancer cells (FIG. 35) were treated with orwithout WZB 117 for 48 hr, and then measured for their respectiveviability rates with the MTT assays. Mock-treated cells served ascontrols (100% viability) for comparison. Noncancerous NL20 and MCF12Acells were treated the same way for comparison. These results suggestthat compound WZB-117 has potential to serve as an anti-cancer agent.FIG. 36 suggests that compound WZB-117 exhibits significantly morecytotoxicities towards cancer cells than towards non-cancerous (i.e.,“normal”) cells.

Examples

General Experimental Protocols

Experimental Protocols. Chemical and Synthetic.

General Scheme for Identifying Improved Basal Glucose TransportInhibitors

Identification of improved small molecule anticancer agents that act asinhibitors of basal glucose transport is outlined in FIG. 24. Upondesign and synthesis of potential basal glucose uptake inhibitors, thecompounds will be examined in both a basal glucose inhibition assay andan apoptosis assay at a single concentration. Compounds that exceed abaseline value will be further examined and IC₅₀ values determined. Theresults of these assays will be used to guide the design of additionalagents.

Pharmacological Evaluation of Inhibitors of Basal Glucose Transport

All compounds will be evaluated in a series of pharmacological assaysdesigned to ascertain the ability of the compounds to both inhibit basalglucose uptake and kill cancer cells. Initial evaluation of compoundswill focus upon elimination of compounds that are unstable in serum orlack appropriate activity.

Compounds will be assayed for their ability to inhibit basal glucoseuptake. All compounds will be tested at a concentration of 10 μM firstand their inhibitory activity on basal glucose transport will bemeasured and compared to that of WZB-27. Cell lines H1299 (lung cancer)and MCF-7 (breast cancer) grown in 24-well cell culture plates will betreated with or without the compounds for 10 min before the glucoseuptake assay. Cellular glucose uptake will be measured by incubatingcells in glucose-free RPMI 1640 buffer with 0.2 Ci/mL [³H]2-deoxyglucose(specific activity, 40 Ci/mmol) for 30 min in the absence and presenceof compounds. After removal of the buffer, the cells are washed withice-cold PBS, cells will be lysed and transferred to scintillation vialsand the radioactivity in the cell lysates will be quantified by liquidscintillation counting. Only those compounds that show comparable orstronger inhibitory activity than that of WZB-27 will be selected forthe next assay. Known glucose transport inhibitors Fasentin (IC₅₀≥50 μM)and apigenin (IC₅₀≥60 μM) will be used as positive controls forcomparison.

Initial assays will also determine the ability of these compounds tokill cancer cells and to leave normal cells untouched. Those compoundsthat pass the criteria of the glucose uptake assay for inhibitoryactivity of basal glucose transport described above will be tested in asubsequent cell killing (viability) assay. Compounds will beindividually added to H1299 (and its normal cell counterpart NL20) andMCF-7 (and its normal cell counterpart MCF-12A) cells grown in 24-wellplates at a concentration of 30 μM and incubated at 37° C. in a cellculture incubator for 48 hrs. After incubation, the cells in each wellwill be measured for its viability by an MTT assay.

Further investigation of compounds will be conducted pending that thecompounds of interest inhibit glucose uptake greater than WZB-27, causea decrease in cancer cell viability of at least 50%, and cause no morethan a 20% decrease in normal cell viability. Further assays forcompounds meeting the minimal initial screening requirements willinclude determination of an IC₅₀ for glucose uptake inhibition and anEC₅₀ for cell killing by including multiple concentration assay pointssuch as 0.1, 0.3, 1, 3, 10, 30, and 50 μM.

Synthesis of More Potent and Selective Inhibitors of Basal GlucoseTransport

The general goal is to generate more potent and selective inhibitors ofglucose uptake. These compounds should have a lower molecular weight,improved water solubility, and good stability. All compounds will beprepared on a 15-20 mg scale and will be purified to >90% purity asanalyzed by HPLC, LCMS and ¹H/¹³C NMR. Each compound will be stored in abar-coded vial as a 50 mM solution in DMSO. This scale will provideample material for initial screens, as well as follow-up screening ifnecessary.

As shown in FIG. 25, lead compounds (WZB-115, WZB-117, and WZB-118) havefour distinct molecular areas of interest, the central aromatic core,the pendant aryl groups, the linker, and the substitution on the centralaromatic core. Each of these four areas will be examined in order todetermine its significance and to develop more potent analogs. Initialfocus will be placed upon the development of non-hydrolyzable linkersthat retain the activity of the parent ester. This will be key forcarrying out in vivo efficacy studies. Concurrently, the replacement ofthe pendant phenol with bioisosteric replacements will be examined in aneffort to improve bioavailability. The substitution and identity of thecentral aromatic core will also be examined in an effort to improvepotency.

Linker Modifications

Contemplated herein are a series of analogs (FIG. 26) in which thelinker between the central aromatic core and the pendant aromatic ringshas been modified. These investigations into modifications of the linkerare focused on the replacement of the potentially labile ester linkage.These studies are significant in that the linker may have a profoundinfluence on the conformational relationship between the centralaromatic core and the pendant aromatic ring. In addition morehydrolytically stable analogs will be of great utility in in vivostudies. Three analogs with all carbon linkages between the corearomatic ring and the pendant aromatic ring (6, 7, 8) will be prepared.Sulfur-linked amalogs will also be prepared, which will include asulfide, sulfoxide, and sulfone linker (9, 10, 11). An ether analog withan oxygen linkage between the core aromatic ring and the pendantaromatic rings (12) will be prepared. Amide-linked analog 13 and twobicycle-linked derivatives (14, 15) will be prepared. All of theseanalogs are based upon the WZB-117 structure. This core has been chosenbased upon the activity of the parent compound and the ease of synthesisbased on the WZB-117 core relative to the WZB-27 core.

Contemplated herein are the energy-minimized structures of the proposedanalogs are shown in FIG. 14. Several analogs stand out in theirsimilarity to the parent WZB-117. These include alkene 7, sulfoxide 10,sulfone 11, and amide 13. All of these analogs have the pendant aromaticrings on opposite sides of the core aromatic ring. As might be expectedalkane 8, sulfide 9, and ether 12 share strong similarities. The highlyflexible nature of these analogs suggest that they can adopt a widevariety of conformers including that of the active WZB-117. Alkyne 6 andbenzimidazole 14 are similar as they share a relatively planar overallstructure. Dioxin 15 is unique, having a single well-definedconformation with both pendant aromatic rings on the same side of thecore aromatic ring. Obviously, the alkyne and alkene derivatives with avery rigid linker provide unique structures unlike any of the others.The synthesis and assay of this set of analogs will provide informationon optimal conformations and determine appropriate hydrolytically stablelinkers.

The first set of analogs contemplated herein will contain a carbonlinkage between the pendant hydroxyphenyl ring and the core fluorophenyl ring and will be prepared using a single synthetic sequence.Starting from the known dibromo fluorobenzene 16, two Sonagashiracouplings with terminal alkyne 17 will be achieved to provide an alkynelinked intermediate. Alkyne 17 can be prepared from the requisitealdehyde or via a Sonagashira coupling of the bromide. Deprotection ofthe hydroxyl group with acid should provide target analog 6. The alkynewill then be partially reduced to provide cis olefin analog 7. Finally,a complete hydrogenation of the olefin will be effected with H₂ and Pdto provide carbon-linked analog 8.

Also contemplated herein are a series of sulfur-linked analogs,including compounds 10 and 11, which will provide bioisosteric andhydrolytically stable analogs of the parent esters. Additionally, thethree analogs (9-11) will alter the acidity of the phenol groups. Thesynthesis of this series starts with the dibromination of commerciallyavailable fluoride 18. The dibromide will then be used to alkylatethiophenol 19 to provide the first analog. Two sequential oxidationswill then provide the sulfoxide (10) and the sulfone (11).

The synthesis of the ether-linked derivative starts with thedialkylation of diol 20 with benzyl bromide (21) to provide a protectedbis ether. Bromide 21 can be readily prepared via thereduction/bromination of the corresponding acid. Deprotection of thephenolic hydroxyl groups with acid will then provide the targetether-linked analog 12.

The amide derivative provides a conformationally restricted analogrelative to the parent ester as well as providing a more hydrolyticallystable derivative. The synthesis is quite straightforward and simplyrequires the acylation of commercially available diamine 23 with therequisite acid. Removal of the MOM group will provide analog 13.

Dioxin derivative 14 will provide a highly conformationally restrictedanalog. As shown below, this compound will be prepared via acondensation of α-bromoketone 27 and diol 20. Bromide 27 can be preparedvia the coupling of Weinreb amide 24 with Grignard reagent 25. This willprovide ketone 26, which can be readily brominated to provide keyα-bromoketone 27. Condensation of 27 with diol 20 followed bystereoselective reduction of the intermediate oxonium ion provides thecis-disubstituted dioxin 14.

Benzimidazole analog 15 provides an alternate conformation relative todioxin 14. In addition the benzimidazole ring should provide improvedwater solubility relative to the parent WZB-117. Compound WZB-117 has aC log P of 2.96 (lower numbers indicating greater water solubility)while analog 15 has a C log P of 2.72. Again, this compound should behydrolytically stable. Diamine 22 will be condensed with aldehyde 28 toprovide substituted benzimidazole 29. Acylation followed by removal ofthe benzyl protecting groups should provide the target compound 15.

This set of compounds will provide an initial structure activityrelationship for glucose uptake inhibition relative to the linker group.Several analogs have been proposed that address the stability of theester group as well as the conformation of the pendant aromatic rings.These analogs represent only the initial targets for modification of theester linkage and further modifications will be made to extend thesestudies as warranted by the pharmacological activity of this set.

Also contemplated herein are the following compounds, wherein R isselected from the group consisting of H, Me, Et, and iPr:

Pendant Bioisosteric Replacements

The 3-hydroxyphenyl group has been identified as the optimal pendantaromatic group. Phenols are known to often have poor bioavailability andshort duration of activity due the facile metabolism, conjugation andexcretion of this group. Given the need to develop more metabolicallystable analogs, a series of phenol bioisosteres will be examined. Dozensof such bioisosteres have been reported in the literature.

Contemplated herein are two general sets of analogs, the acetamido groupand a series of heterocyclic bioisosteres. 3-Aminobenzoic acid will beconverted to an acetamide, methansulfonamide, and a urea and then coupleto diol 20 to provide analogs 31, 32, and 33, respectively. Anadditional analog (34), based on the replacement of a phenol with ahydroxymethyl group as seen in the 03 adrenergic blocker albuterol, willalso be prepared. Four different heterocyclic derivatives (35-38) willbe prepared by coupling the commercially available heterocycliccarboxylic acid with diol 20. This set of compounds will provideinformation on the ability to replace the phenolic group withpotentially more metabolically stable moieties.

Also contemplated herein are the following compounds, wherein X isselected from the group consisting of H, 3-Cl, 3-F, 3-CN, 4-F, 4-CN,4-NO₂, 4-SO₂Me, and 4,5-Cl₂:

Optimization of the Central Aromatic Core

Also contemplated herein is a third group of analogs, in which thecentral aromatic ring from WZB-117 and WZB-118 will be modified. Thisstudy will allow for the optimization of the activity of WZB-117 andWZB-118 analogs through changes in the substitution (i.e. the F or Clgroup) on the aromatic ring. The introduction of a chloro- orfluoro-group to the aromatic core and the removal of one of the benzoylgroups has resulted in improved potency and a lower molecular weight. Ithas been demonstrated that the 3-fluoro and 4-chloro derivatives (whencounting starting with the furthest O-benzoyl group) have the bestactivity. In terms of generating additional analogs, this means that thediols shown in FIG. 29 will be critical to further scaffold elaboration.A Topliss tree type approach has been employed to generate many of thesederivatives. These derivatives will be acylated with acid chloride 23and then deprotected as shown previously. These compounds arecommercially available or may be easily prepared from a commerciallyavailable precursor by one who is skilled in the art. Compounds 39, 42,46, and 43 are commercially available. Nitriles 40 and 44 can beprepared via an oxidative conversion of the corresponding aldehyde tothe nitrile. Nitro derivatives 41 and 45 may be prepared by nitrationwith a zeolite supported copper nitrate reagent.

This set of compounds will provide valuable insight into optimalsubstitution on the central aromatic ring and provide directions for thesynthesis of generation 4 and 5 analogs. This set of derivatives is asmall sample of potential analogs that will be synthesized as a resultof positive biological outcomes from generation 2 analogs, and furthergenerations of analogs may be synthesized based upon the combination ofresults from several specific modifications.

Experimental Protocols. Biological

Compounds. Powders of compounds were stored at −20° C. and solutionswere freshly prepared before each experiment. Compounds were dissolvedin DMSO to make 10 mM stock solution. In most studies, 30 μM WZB-27 and10 μM WZB-115 were used for cell treatment.

Cell lines and cell culture. Human non-small cell lung cancer (NSCLC)cell lines H1299 and A549, human duct epithelial breast cancer MCF7, andhuman non-tumorigenic NL20 lung and MCF12A breast cells were purchasedfrom ATCC. H1299, A549 and MCF7 cells were maintained in Dulbecco'sModified Eagle Medium (DMEM) with 10% fetal bovine serum. NSCLC celllines A549, H358, H226, and H1650 are grown in Ham's F12K containing 10%FBS. NL20 cells were maintained in a Ham's F12 medium, supplemented with0.1 mM nonessential amino acids, 0.005 mg/ml insulin, 10 ng/ml epidermalgrowth factor, 0.001 mg/ml transferring, 500 ng/ml hydrocortisone, and4% fatal bovine serum. MCF12A cells were cultured in a 1:1 mixture ofDMEM and Ham's F12 medium, with 20 ng/ml human epidermal growth factor,100 ng/ml cholera toxin, 0.01 mg/ml bovine insulin, 500 ng/mlhydrocortisone and 5% horse serum. All cells were grown at 37° C. in ahumid atmosphere with 5% CO₂. Cells were treated with compound WZB-27 orWZB-115 at concentration of 30 μM or 10 μM, respectively, for 24 or 48hours. Untreated cells were used as control.

Cell lysate preparation and Western blot analyses. Protocol 1. Cellswere harvested from plates, re-suspended with 3× sample buffer, andboiled for 5 min. Approximately 50 μg of protein extract was loadedafter protein concentration measurement by Pierce bicinchoninic acid(BCA) protein assay (Pierce Biotechnology, Inc. Rockford, Ill.). Sampleswere run on a 10% Bio-Tris NuPAGE gel (Invitrogen) and transferred to apolyvinylidene difluoride membrane (PVDF, Biorad). The membrane wasincubated with antibodies specific to p53, or PARP, or β-actin. Specificprotein bands were visualized after the development of film. Antibodiesfor p53 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,Calif., USA). The PARP antibody and β-actin antibody were purchased fromCell Signaling Technology, Inc. (Danvers, Mass., USA).

Cell lysate preparaztion and Western blot analyses. Protocol 2. Lysatesfrom cells are prepared by NP40 lyses. Samples are boiled in equalvolume of 2×SDS sample buffer, and separated on 8% polyacrylamide gels.After semi-dry transfer to supported nitrocellulose membranes, the blotsare probed with monoclonal antibody to GLUT1 from R&D systems. Theproteins are detected by using an enhanced chemiluminescence assaysystem from Amersham Biosciences.

Real time RT-PCR protocol. Total RNA was isolated using Trizol®(Invitrogen, Carlsbad, Calif.). In order to eliminate any carryover ofgenomic DNA, total RNA was treated with DNAse using the DNA-free kit(Ambion, Austin, Tex.). cDNA was synthesized from total RNA using theAdvantage RT for PCR (BD Biosciences, Palo Alto, Calif.). One mg of thetotal RNA was used in a 50 μl reaction mixture with the random hexamerprimer. Real time primers and TaqMan® probes for GAPDH were purchasedfrom Biosource (Camarillo, Calif.), and were used according to themanufacturer's instructions. Three ml of cDNA template were used in 25μl of real time PCR reaction with ABI TaqMan® Universal Master Mix(Applied Biosystems, Branchburg, N.J.). The GLUT1 detection is done withSybr® green dye and the Quntitect Sybr®Green kit according to themanufacturer's instructions, using 1 μl of cDNA template in a 25 μlreaction volume.

Glucose uptake assay. Cancer cells are treated with or without thecompounds for 10 min before the glucose uptake assay. Cellular glucoseuptake will be measured by incubating cells in glucose-free RPMI 1640with 0.2 Ci/mL [³H]2-deoxyglucose (specific activity, 40 Ci/mmol) for 30min in the absence and presence of compounds. After the cells are washedwith ice-cold PBS and lysed, the cell lysates will be transferred toscintillation counting vials and the radioactivity in the cell lysatesis quantified by liquid scintillation counting.

Cell cycle analysis. After being treated by compounds or medium with lowconcentration of glucose, cells were harvested, washed with cold PBS,and re-suspended in 70% cold ethanol. After an overnight fixation,ethanol was removed and cells were treated with propidium iodide,DNase-free RNase A, and PBS mix (4:1:95) for 30 min at 37° C. The DNAcontent was analyzed by flow cytometry (FACS, BD). Modfit software(Verity Software House) was used to calculate the percentage of cells ineach phase of the cell cycle. Each sample was repeated three times.

Cell viability assay. MTT assays are performed by the followingwell-established method. In a 96 well tissue culture plate 10,000 cellsare plated in each well. The cells are incubated in presence or absenceof the compounds for 18 h.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) isdissolved in PBS (10 mg/ml) and filter sterilized. Three hours beforethe end of the incubation 20 μL of MTT solution is added to each wellcontaining cells in a 96-well plate. The plate is incubated in anincubator at 37° C. for 3 h. Media is aspirated gently and 200 μL ofDMSO is added to each well to dissolve formazan crystals. The absorbanceis measured at 550 nm for cell viability.

Proliferation assay. MCF-7, H1299 and H1650 cells are plated ontopoly-d-lysine (Sigma) coated 8-well glass chamber slides (10,000 cellsper well). The cells are incubated with the compounds for 24 h or 48 hr.The cells are fixed and stained using 5-Bromo-2′-deoxyuridine labelingand Detection kit from Roche according to manufacturer's protocol.

Study for anticancer synergistic effect. H1299, and MCF7 cells weregrown in 96-well plates. Cells were treated with either cisplatin ortaxol at their IC₅₀s in presence or absence of compound WZB-27 (30 μM)or WZB-115 (10 μM) at 37° C. for 48 hours. Viability of the treatedcells was measured by the MTT cell proliferation assay.

Apoptosis assay. MCF-7, H1299 cells are plated onto poly-d-lysine(Sigma) coated 8-well glass chamber slides (10,000 cells per well). Thecells incubated with the compounds of various concentrations for 24 h.Cells without compound treatment serve as controls. After 24 h ofincubation cells are fixed and stained according to manufacturer'sinstructions using Promega's DeadEnd Colorimetric TUNEL system. Thecooperative effect of drugs will also be evaluated by adding 5 μM ofcisplatin or paclitaxel or 10 μM gefitinib by the same procedure.

Immunofluorescence. GLUT1 monoclonal antibody will be purchased from R&DSystems Inc (Minneapolis, Minn.). Cells are plated onto poly-d-lysine(Sigma) coated 8-well glass chamber slides (10,000 cells per well) forimmunostaining. Cells are fixed in 3.5% paraformaldehyde for 25 min,permeabilized in 0.2% Triton X-100/PBS for 5 min, and blocked in 5%normal goat serum in PBS at room temperature for 1 h. Primary antibodyincubation is performed overnight at 4° C. After washing, secondaryantibody incubation is performed with goat anti-mouse IgG AlexaFluor-488 for 30 min at room temperature. DAPI is detected usingVectashield Mounting Medium with DAPI (Vector Laboratories, Inc.).Staining by secondary antibodies only will be used as negative controls.Slides are observed by fluorescence microscopy using fluorescentmicroscope (40×/0.75 numerical aperture) with a camera.

Statistical analyses. Samples of same experimental conditions are intriplicate or more. Each experiment is repeated at least once (animalstudies are exceptions). Data will be reported in mean±standarddeviation or standard error of means. Data will be analyzed usingunpaired Student t-test and with one-way or two-way ANOVA with Turkey'spost-hoc test depending on the nature of the assays. Significance levelwas set at p≤0.05.

Experimental Protocols. Animal Studies

The ability of compounds to inhibit/reverse tumor growth in nude mice,as well as the clinical safety of the compounds will be determined. Onlycompounds that meet the following criteria will be considered for animalstudies; IC₅₀ of <10 μM in the glucose uptake inhibition, and an EC₅₀ of<10 μM in cell killing assays as well as significant less killing innormal cells.

For the anticancer efficacy and safety animal study, each selectedcompound will be use to treat nude mice with cancer grown from H1299(lung cancer) and MCF-7 (breast cancer) cells. Five millions of cancercells will be injected subcutaneously into the flank of each of 15 nudemice. The tumor cell-injected mice will be randomly split into threegroups: five for compound treatment, five for drug (e.g. WZB-117)treatment (positive controls) and another five receive vehicle (solvent)treatment. After tumors become palpable and visible (˜2-3 weeks), thecompound treatment will begin. A molar concentration of EC₅₀ will bechosen for each compound for the treatment. Compounds will be dissolvedin DMSO or other compatible solvent.

The IP injection of compounds will be performed 3 times a week for 3-5weeks depending upon tumor growth rates. Tumor sizes will be measuredwith calipers twice a week and recorded as W×L×H=volume in mm³ andcompared to those of tumors on non-compound injected control mice. Bodyweight of the mice is measured once a week. In order to determine howcompound treatment affects blood glucose levels, blood glucose will alsobe measured immediately prior to the compound injection and 1 hr afterthe injection and the blood glucose levels will be compared to those ofthe non-compound injected mice. The compound treatment lasts 3-5 weeksuntil the tumors grown in the untreated mice become large (>5% but <10%of the body weight). The animal study will be carried out and terminatedin accordance to the rules and regulations of NIH and of our universityIACUC. Tumor-bearing mice will be euthanized at the end of the study,according to the related rules by NIH and DOA. The average size of thetumors in the treated groups will be compared to the untreated controlgroup to show treatment efficacy and statistical differences.

The best compound(s), based on combined consideration of anticancerefficacy and toxicity (primarily its effect on blood glucose levels andbody weight changes and/or other unexpected side effects), will bechosen for a larger scale animal study described below.

Investigation of the Molecular Mechanism of Action of Basal GlucoseTransport Inhibitors

Anticancer Activities in More Cancer Cell Lines

Contemplated herein is a cancer cell line screen including multiple lungand breast cancer cell lines that will be tested with compounds todetermine their anticancer activities via the MTT assay to demonstratethat the compounds' cancer cell killing activity is an activity that iscell line-independent. H1299 cells are derived from non-small cell lungcancers (NSCLC). Other NSCLC cell lines such as A549, H358, H226, andH1650 will also be tested. Similarly, the compounds will also be testedin breast carcinoma cell line T47D along with MCF-7 to determine if thecompounds exhibit similar anti-cancer activity in the additional breastcancer cell lines. Normal cells of same tissues (NL20 for normal lungtissue and MCF-12A for normal breast tissue) will be included in thestudy to establish compounds' increased cytotoxicity and killing tocancer cells than to their normal counterparts. To correlate inhibitionof basal glucose transport with cytotoxicity of the compounds, glucoseuptake rates of cancer and normal cell lines will be measured andcompared using the glucose uptake assay. The cancer cell lines areexpected to exhibit higher glucose uptake rates than their normal cellcounterparts due to their higher energy needs. Increased cell killing incancer cells opposed to normal cells may be due to the inhibition ofbasal glucose transport that is crucial for cancer cell proliferationand survival. This study will also further strengthen the notion thatthe basal glucose transport is the target of the anticancer action ofthe compounds disclosed in this patent and related publications. Theenzymatic activity of hexokinase, the first enzyme involved inglycolysis, will also be measured with standard assays in the compoundtreated cells using untreated cells as a control to determine howglycolysis is affected by the compound treatment in these cancer cells.

In addition to the cell lines described, select compounds will besubmitted to the Developmental Therapeutics Program's NCI 60-Cell LineScreen.

Also contemplated herein is the theory that the basal glucosetansporters disclosed will potentiate the chemotherapeutic effects ofother anti-cancer agents. In order to determine if the compounds canpotentiate anti-cancer activity of other anticancer drugs, 5 μM ofcisplatin or paclitaxel or 10 μM gefitinib will be added to H1299 andMCF-7 cells in the absence and presence of the compound for 48 hrs.After incubation, cell viability will be determined by both MTT and thecell proliferation assays (see general procedures at the end of thissection for details). (Drug+compound) treated samples will be comparedto those samples treated by drugs alone. Increased cell death in the(drug+compound) treated samples indicates that the compound couldpotentiate the anticancer activity of the drugs.

Additional Receptor Binding Assays

Contemplated herein are a series of receptor binding assays which willbe used to determine the action target of the basal glucose transportinhibitor disclosed in this application. In order to investigate theaction target of the compound, a binding competition study will becarried out. Anti-GLUT1 antibodies will be added to H1299 or MCF-7 cellsin the absence and presence of increasing amount of the compound at 37°C. for 1 hr. After co-incubation, unbound antibodies and compound willbe removed by washing. The treated cells will be incubated with asecondary antibody (goat anti-mouse IgG Alexa Fluor-488) that interactswith the bound anti-GLUT1 antibodies. A “chromogenic” reaction will beperformed after the secondary antibody binding. The intensity offluorescence generated by the bound secondary antibodies should beproportional to the GLUT1-bound primary antibodies. The intensity of thefluorescence of differently treated cells will be quantified andcompared. The decrease of the intensity in the antibody/compound treatedsamples suggests that the presence of the compound decreases the bindingof GLUT1 antibodies to GLUT1 and further strongly suggests that thecompound bind to GLUT1 located on the cell membrane. On the other hand,if no competition is found, it does not necessarily mean that thecompound does not bind to GLUT1. It may also mean that the compoundbinds to a place on GLUT1 that is different from the binding site ofanti-GLUT1 antibody.

To further determine how compounds work, the anti-GLUT1 antibody of afixed concentration will be added to cancer cells in a glucose uptakeassay in the absence and presence of the compounds of variousconcentrations. In a similar study, the compound concentration can befixed and the antibody's concentration can be varied. These glucoseuptake assays are to determine whether the basal glucose transportinhibitory activities of anti-GLUT1 antibody and the compounds areadditive or synergistic to each other. If the activities are additive toeach other, it may suggest that these two agents act, probably but notnecessarily, on the same target. If the activities are synergistic, itis more likely that these two molecules act on different targets. It isalso possible that the effects may not change or even decrease whencompounds are added with GLUT1 Ab.

Contemplated herein is a method to directly show the binding of thecompound to GLUT1, which will be accomplished by the inclusion of afluorescent tag as a moiety on any lead compounds. It is anticipatedthat more potent and selective analogs will be identified prior to thepreparation of fluorescent tracers. Two approaches will be used toidentify the necessary fluorescent probes. As an illustrative example wewill show a synthesis based on the current lead compounds. In the firstapproach we will replace a pendant aromatic ring with a fluorescent tag.Depending upon the SAR for these compounds this could be the optimalapproach in that we can use fluorescent tags similar to the pendantaromatic rings. Thus a significant change in affinity to the biologicaltarget would be decreased. As shown below, we would monoprotect triol 1,and then introduce two esters onto the free hydroxyl groups. Theprotected hydroxyl would be deprotected to provide 48. The fluorescentcoumarin 49 would be coupled to provide the target fluorescent probe 50.

A second approach will be to simply label an active compound with afluorescent tag. For example we would couple WZB-113 with any of anumber of commercially available fluorescent carboxylic acids(flRCOOH=49 or rhodamine, or carboxynaphthofluorescein). Based on theactivity of WZB-113 relative to WZB-117 only one of the two hydroxylgroups is likely involved in significant non-covalent interactions. Thuswe can use one of these hydroxyl groups to attach the fluorescent tag.

All fluorescently tagged compounds will first be evaluated for theirability to both inhibit basal glucose transport and induce apoptosis. Ifthe fluorescent analogs do not act in a similar manner to the parentcompound new derivatives will be prepared.

Varying concentrations of the fluorescent compound, will be used toincubate with GLUT1 pre-bound to the bottom of a 96-well plate with aprotocol similar to that previously used for insulin receptor binding ofthe polyphenolic compound PGG. After an overnight incubation at 4° C.with shaking, the unbound compound will be washed off. The fluorescenceintensity of different samples will be measured with a 96-well platereader (SPECTRA Max M2, software: SoftMax Pro). A GLUT1 bindingsaturation curve can be generated from intensities corresponding todifferent compound concentration. The GLUT1 binding affinity (K_(a)) canalso be generated from this binding experiment. A binding displacementcurve can also be generated from an assay in which an increasing amountof regular compound is added to GLUT1 pre-bound to 96 well plates whilefluorescent compound is kept at a fixed concentration. At higherconcentrations, the regular compound will compete with fluorescentcompound for the same binding site on GLUT1, reducing the fluorescentintensity proportionally to the increased concentrations of the regularcompound. The binding affinity of the compound (K_(d)) can be derivedfrom the displacement curve using computer software.

Relationships and Signaling Pathways Linking Inhibition of Basal GlucoseTransport and Induction of Apoptosis in Cancer Cell Lines

Although it has been shown that addition of compounds leads toinhibition of basal glucose transport inhibition and apoptosis (cancercells), the cause-effect relationship between the inhibition andapoptosis has not been established. To determine the relationship, cellsamples treated with anti-GLUT1 antibody will be used as a positivecontrol. Since the anti-GLUT1 antibody has only one target, GLUT1, theapoptosis induced by the addition of the antibody is the “direct effect”caused by the antibody. In this study, the apoptosis induced by thecompounds will be compared side by side with the apoptosis induced bythe antibody. The parameters compared include: 1) onset time of theapoptosis induced; 2) dose responses of apoptosis; 3) apoptosis inducingmechanism. It has been disclosed that the apoptosis induced by compoundsWZB-25 and WZB-27 is p53-independent (FIG. 24A). If the apoptosisinduced by the antibody is also p53-independent (p53 is notsignificantly changed by the antibody treatment), then this evidencewould support the fact that these compounds induce apoptosis in cancercells using a mechanism similar to that of the antibody. If theapoptosis induced by the antibody is p53-dependent (p53 is activated),it indicates that the antibody uses a mechanism in apoptosis inductiondifferent than the one used by the compounds. This will strongly suggestthat compounds inhibit a different target than GLUT1.

Also contemplated herein is a proteomic approach to study the linkbetween inhibition of basal glucose transport and induction ofapoptosis. The selected compound will be used to treat H1299 and MCF-7cells for 24 hrs with untreated cells as negative controls. Totalproteins will be isolated from the treated and the untreated cells.Protein samples will be treated with protease inhibitor, TBP and samplebuffer which contains urea, thiourea, and CPHAS for 2 hours. Then IAAwill be added. Twenty min later, IAA will be added to the samples again.After treatment, samples will be loaded to strips and the strips withsamples will be kept in room temperature for 2 hours before they areplaced in the first dimension gel electrophoresis instrument for thefirst dimensional protein separation. After finishing the firstdimensional protein separation, the strip will be loaded on SDS-PAGEgels for the secondary protein separation. After the separation, gelswill be fixed overnight with fixing buffer containing ethanol, aceticacid, and SDS. Then the gels will be washed with washing buffercontaining acetic acid and SDS before they are stained with sypro orangefor 2 hours for spot detection. The 2-D gel results will be analyzedwith software of PDQuest.

Protein spots will picked automatically under control of a camera andtransferred to a 96-well microtiter plate with holes in the bottom. Themicrotiter plate with all gel spots will be transferred to an automaticdigester (Tecan GmbH) to wash the gel pieces, digest the protein withtrypsin at 50° C. for 2 hrs. The digest will be done with 2 mMammoniumhydrogen carbonate buffer (pH 7.8) to reduce the salt contentand the and 0.5 μL of the extracted peptides are automatically spottedon a MALDI target, whereas the remaining 20 μL are stored in amicrotiter plate. Usually about 90% of all protein spots can be alreadyidentified from the MALDI-TOF/TOF mass spectra by combining a peptidemass fingerprint with the tandem mass spectra of the top five peptidesignals. Alternatively, the sample stored in the microtiter plate can beanalyzed my nanoRP-HPLC-nano-ESIQqTOF-MS/MS, which usually gives abetter sequence coverage and often a better confidence level. Overallproteins can be identified at the 100 fmol level.

Twenty up-regulated protein spots and twenty down-regulated spots on asingle gel, as compared to the untreated control gel, will be selectedfor MS analysis and amino acid sequence determination. Considering theintrinsic variations of the system, only those spots that are eitherup-regulated by 2-fold or more or down-regulated by 2-fold or more willbe chosen. After the amino acid sequence of the N-terminus of a proteinis determined, the identity of the protein will be uncovered bycomparing the sequence with the protein sequences in the data bank. Byknowing which proteins are up-regulated or down-regulated by thecompound treatment, these proteins will be categorized into differentgroups and different metabolic and/or signaling pathways, which shouldenable us to identify pathways that are activated or inactivated by thecompound, providing clues for how the inhibition of basal glucosetransport leads to eventual induction of cancer cell apoptosis.

In Vivo Anticancer Studies

Although the compounds' anticancer activity in cancer cell lines hasbeen established in preliminary studies and in a recent GLUT1 antibodystudy in multiple cancer cell lines, it has also been tested in animalmodels, which is an intermediate step for moving cancer research fromlaboratory to clinics. Secondly, inhibiting basal glucose transport mayinduce hyperglycemia in the treated animals. To address the question ofin vivo efficacy and safety, a compound selected based on its improvedIC₅₀ (basal glucose transport), EC₅₀ (cancer cell killing), andmaintained/improved target selectivity (improved killing in cancer cellswithout increased killing in normal cells), as well as anticancerefficacy and safety findings from animal study described above will beused in this animal study.

The objectives of the proposed animal study are to determine if thecompound treatment reduces cancer growth and if the compound treatmentis safe to the tumor-bearing mice. The effective and safe doses will bechosen based on cell killing assays on cancer cell lines and tolerablecytotoxicity in the normal counterparts of the cancer cells tested aswell as in a short term pilot animal study similar to the one describedabove, in which the cell study-determined compound dose and 2× and 4×doses will be tested in nude mice (3 per group). In the pilot study, thecompound will be administered to mice once a day for five days and thecompound-injected mice will be monitored for signs of side effects(hyperglycemia immediately after compound treatment and with time,reduction and difficulty in movement, loss of body weight). A safe dosewill be selected and an animal study will be performed as follows:

Because human cancer cell line(s) will be used, immune-deficient nudemice will be used. A total of 30 nude mice will be used, 10 mice pergroup. These mice will be randomly selected into each group. Group 1 isthe negative control group, which will be inoculated with cancer cellsbut without receiving subsequent compound treatment; Group 2 will be thelow dose compound treatment group and group 3 will be high dose compoundtreatment group. Five million cells of either H1299 or MCF-7 cell lineswill be injected subcutaneously (SubQ) into the flank of nude mice. Wedecided to use H1299 NSCLC and MCF-7 cells as our cancer models werebased on these considerations: (a) although the inhibitors showedanticancer activity in all the cancer cell lines we tested, they showedeither the higher anticancer activity or higher cancer cell: normal cellkilling ratios or both among all cell lines tested, (b) NSCLC and breastcancers are two cancers that have been considered major targets ofcurrent cancer research and therapeutic treatment. In addition, GLUT1has been found over-expressed in these two cancer types.

The compound treatment will start when tumors become palpable andvisible. The compound at a concentration of 10 mg/kg of body weight willbe intraperitoneally (IP) injected, one injection for every other day(except the weekends) for the entire study. The negative control micewill be injected exactly the same way but with vehicle (the solution inwhich the compound is dissolved). Tumor size will be measured twice aweek with calipers, and dimensions of length, width and height of thetumors will be measured and recorded (L×W×H) as tumor volumes. Thecompound treatment lasts 3-5 weeks until the tumors grown in theuntreated mice become large (>5% but <10% of the body weight). Theanimal study will be carried out and terminated in accordance to therules and regulation of NIH and our university IACUC. Tumor-bearing micewill be euthanized at the end of the study according to the relatedrules by NIH and DOA. The average size of the tumors in the treatedgroups will be compared to the untreated control group to show treatmentefficacy and statistical differences. The tumor size will also becompared between the high dose group and the low dose group to show thedose response of the treatment. The food intake, body weight, as well asblood glucose levels will also be measured twice a week to monitor theanimal health and to compare these health parameters with the untreatedcontrol group. At the end of the animal study, after animal euthanasia,tumors will be removed from the compound treated mice and from theuntreated control mice. Total proteins will be isolated from the tumorsand their respective p53 and caspase 3 will be measured to determine ifmore apoptosis is induced in the compound treated mice and if there isany change in the activated p53. This is to determine if the in vivoanticancer mechanism is the same as observed in cancer cell lines.

Animal Tumor Treatment Study. Protocol

1. Study from November 2009 to January 2010, 10 weeks; 2. Nude mice(immunodeficient), ten mice per group; 3. Tumor model—Human lung cancerA549 (NIH recognized and recommended), 5×10⁶ cells injected into theflank of each mouse subcutaneously; 4. Treatment with or withoutcompound WZB-117, PI injection daily for all 10 weeks, dose=15 mg/kgbody weight; 5. Weekly measurements: tumor size, body weight, foodintake; and 6. Other indicators measured: blood glucose, serum insulin,body composition, and blood cell counts. FIGS. 30-34 and Tables 8-11show the results of this study.

TABLE 8 Body mass composition Body Treat- weight Fat Fluid Lean ment (g)g % g % g % Control 29.30 ± 1.55 2.84 ± 0.61  9.74 ± 2.29  1.78 ± 0.386.02 ± 1.07 23.61 ± 1.64 80.54 ± 2.22 WZB117 28.48 ± 0.17 1.05 ± 0.17*3.69 ± 0.61* 1.80 ± 0.12 6.32 ± 0.40 24.40 ± 0.72 85.69 ± 2.40

Minispec data indicated that the difference in body weight between thecontrol and WZB-117 treated group was primarily due to decrease in fattissue in the WZB-117-treated mice. Body mass composition of each mousewas measured by the Minispec NMR Analyzer mq7.5 (Bruker, Billerica,Mass.) after 70 days of compound WZB-117 treatment. Results wereanalyzed by an OPUS program (Bruker).

TABLE 9 Blood cell count analysis of differently treated mice No tumorno PBS + DMSO WZB 117 CBC parameters treatment group treatment grouptreatment group Normal range WBC (K/μl) 10.1 ± 3.3 9.1 ± 1.6 5.9 ± 0.3 1.8-10.7 LYMPH (K/μl)  7.2 ± 2.2  49 ± 1.6 2.0 ± 0.7 0.9-93  RBC (M/μl)10.0 ± 0.3 8.7 ± 1.7 8.8 ± 2.0 6.36-9.42 HGB (g/dL) 15.9 ± 0.5 13.7 ±2.0  13.8 ± 2.6  11.0-15.1 PLT (K/μl) 1522.5 ± 159.7 1427.9 ± 327.0 2214.0 ± 192.3   592-2972

Blood was collected after 70 days treatment through mouse tail veinusing heparinized capillary tubes and then transferred to EDTAcontaining microfuge tubes. Blood was analyzed using Hemavet 950hematology system from Drew Scientific (Dallas, Tex.). CBC analysesindicated that, compared to the PBS-DMSO-treated group, WZB-117 treatedgroup showed reduced counts in WBC, lymphocyte, as well as increasedplatelet count. However, these changes for the treated group were stillin the normal ranges.

TABLE 10 Serum insulin levels in differently treated mice. TreatmentSerum insulin (mg/l) No tumor no treatment 0.971 ± 0.373 PBS + DMSO0.743 ± 0.104 WZB117 0.572 ± 0.319 Normal range 0.5-10

Serum from each mouse was obtained by centrifuging the blood from mousetail vein at 10,000 rpm for 10 minutes and keeping the supernatant.Serum insulin level of each mouse was measured using MercodiaUltrasensitive Mouse Insulin ELISA (Uppsala, Sweden). Compared to thePBS-DMSO-treated group, WZB-117 treated group showed reduced circulatinginsulin in serum. However, the change was still in the normal range.Considering that there was no significant difference in blood glucoselevels between untreated and compound WZB-117 injection group, thereduced but normal serum insulin level indicated the normal function ofpancreas after 70 days treatment of compound WZB-117.

TABLE 11 HPLC analysis of compound stability in human serum Compoundconcentration (%) Time after incubation with serum Compound 0 h 1 h 2 h4 h 8 h 16 h 24 h 48 h WZB-115 100 41.6 18.9 18.4 0.6 — 0 0 WZB-117 10025.1 15.2 10.1 7.7 7.8 5.4 5.0 WZB-141 100 100.3 — 103.5 — — 109.7 87.4WZB-149 100 86.4 50.7 43.9 50.9 65.0 51.0 62.4

A total of 2.5 ml of compound solution was added to 22.5 ml of humanserum and the mixture was incubated at 37 C for various times. The finalconcentration of the compound in serum was 6.8 mM. After incubation, 200ml of acetonitrile was added to the mixture, centrifuged to removeproteins before HPLC analysis. The relative concentration of samples at0h (in serum but without incubation) was arbitrarily assigned a value of100% and all other samples were compared to the 0h samples. Analysisindicated that ether bond-containing compounds WZB-141 and WZB-149 weremuch more stable in serum than ester bond-containing compounds WZB-115and WZB-117.

What is claimed is:
 1. A compound of formula (II):

wherein R₁ is selected from the group consisting of hydrogen, alkyl,benzyl, aryl, and heteroaryl; wherein R₂ is selected from the groupconsisting of hydrogen, alkyl, benzyl, aryl, and heteroaryl; wherein Xis selected from the group consisting of hydrogen, halo, alkyl, benzyl,aryl, heteroaryl, amino, cyano, and alkoxy; and wherein Y is selectedfrom the group consisting of hydrogen, halo, alkyl, benzyl, aryl,heteroaryl, amino, cyano, and alkoxy; or, a salt thereof.
 2. Thecompound of claim 1, wherein R₁ and R₂ are aryl.
 3. The compound ofclaim 2, wherein R₁ and R₂ are independently selected from the groupconsisting of 2-, 3-, and 4-hydroxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-,and 3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-, and3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl, andperhydroxyphenyl.
 4. The compound of claim 3, wherein R₁ and R₂ are3-hydroxyphenyl; X is fluorine; and Y is hydrogen.
 5. A method oftreating cancer, the method comprising: administering to a subject inneed of such treatment a therapeutically effective amount of a basalglucose transport inhibitor compound of formula (II) or apharmaceutically acceptable salt thereof:

wherein R₁ is selected from the group consisting of hydrogen, alkyl,benzyl, aryl, and heteroaryl; wherein R₂ is selected from the groupconsisting of hydrogen, alkyl, benzyl, aryl, and heteroaryl; wherein Xis selected from the group consisting of hydrogen, halo, alkyl, benzyl,aryl, heteroaryl, amino, cyano, and alkoxy; and wherein Y is selectedfrom the group consisting of hydrogen, halo, alkyl, benzyl, aryl,heteroaryl, amino, cyano, and alkoxy; and whereby administration of saidbasal glucose transport inhibitor compound of formula (II) or apharmaceutically acceptable salt thereof to the subject treats saidcancer by inhibiting basal glucose transport in said subject.
 6. Themethod of claim 5, wherein the cancer comprises solid malignant tumors.7. The method of claim 5, wherein the cancer upregulates basal glucosetransport.
 8. The method of claim 5, wherein R₁ and R₂ are independentlyselected from the group consisting of 2-, 3-, and 4-hydroxyphenyl, 2,3-,2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-,and 3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl, andperhydroxyphenyl.
 9. The method of claim 5, wherein R₁ and R₂ are3-hydroxyphenyl; X is fluorine; and Y is hydrogen.
 10. The method ofclaim 5, wherein the basal glucose transport inhibitor compound offormula (II) or pharmaceutically acceptable salt thereof is administeredby at least one of the following methods: oral, topical, intra-arterial,intrapleural, intrathecal, intraventricular, subcutaneous,intraperitoneal, intraveneous, intravesicular, and gliadel wafers. 11.The method of claim 5, wherein the subject is a human.
 12. The method ofclaim 5, further comprising administering to the subject in need of suchtreatment a second cancer drug.
 13. The method of claim 10, wherein thesecond cancer drug is selected from the group consisting ofmethotrexate, doxorubicin hydrochloride, fluorouracil, everolimus,imiquimod, aldesleukin, alemtuzumab, pemetrexed disodium, palonosetronhydrochloride, chlorambucil, aminolevulinic acid, anastrozole,aprepitant, exemestane, nelarabine, arsenic trioxide, ofatumumab,bevacizumab, azacitidine, bendamustine hydrochloride, bexarotene,bleomycin, bortezomib, cabazitaxel, irinotecan hydrochloride,capecitabine, carboplatin, daunorubicin hydrochloride, cetuximab,cisplatin, cyclophosphamide, clofarabine, ifosfamide, cytarabine,dacarbazine, decitabine, dasatinib, degarelix, denileukin difitox,denosumab, dexrazoxane hydrochloride, docetaxel, rasburicase, epirubicinhydrochloride, oxaliplatin, eltrombopaq olamine, eribulin mesylate,erlotinib hydrochloride, etoposide phosphate, raloxifene hydrochloride,toremifane, fulvestrant, letrozole, filgrastim, fludarabim phosphate,pralatrexate, gefitinib, gemcitabine hydrochloride,gemcitibine-cisplatin, gemtuzumab ozogamicin, imatinib mesylate,trastuzamab, topotecan hydrochloride, ibritumomab tiuxetan, romadepsin,ixabepilone, palifermin, lapatinib ditosylate, lenalidomide, leucovorincalcium, leuprolide acetate, liposomal procarbazine hydrochloride,temozolomide, plerixafor, acetidine, sorafenib tosylate, nilotinib,tamoxifen citrate, romiplostim, paclitaxel, pazopanib hydrochloride,pegaspargase, prednisone, procarbazine hydrochloride, proleukin,rituximab, romidepsin, sunitinib malate, thalidomide, temsirolimus,toremifene, trastuzumub, pantiumumab, vinblastine sulfate, vincristine,vorinostat, zoledronic acid, and any combination thereof.
 14. A compoundof formula (I):

wherein R₁ is selected from the group consisting of hydrogen, alkyl,benzyl, aryl, and heteroaryl; wherein R₂ is selected from the groupconsisting of hydrogen, alkyl, benzyl, aryl, heteroaryl, and fluorescenttags; and wherein R₃ is selected from the group consisting of hydrogen,halo, alkyl, benzyl, aryl, heteroaryl, amino, cyano, and alkoxy; or asalt thereof.
 15. The compound of claim 14, wherein R₁ and R₂ are aryl.16. The compound of claim 15, wherein R₁ and R₂ are independentlyselected from the group consisting of 2-, 3-, and 4-hydroxyphenyl, 2,3-,2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-,and 3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl, andperhydroxyphenyl.
 17. The compound of claim 14, wherein R₁ is aryl andR₂ is a fluorescent tag.
 18. The compound of claim 17, wherein R₁ isselected from the group consisting of 2-, 3-, and 4-hydroxyphenyl, 2,3-,2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxyphenyl, 2,3,4-, 2,3,5-, 2,3,6-,and 3,4,5-trihydroxyphenyl, 2,3,4,5- and 2,3,4,6-tetrahydroxyphenyl, andperhydroxyphenyl; and wherein R₂ is selected from the group consistingof coumarins, dansyl, rhodamine, fluorescein, carboxynaphthofluorescein,and fluorescent proteins.
 19. The compound of claim 14, wherein R₁ andR₂ are 3-hydroxyphenyl; and R₃ is hydrogen.